Brain Energetics and Neuronal Activity: Applications to fMRI and Medicine

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Author: Robert G. Shulman | Douglas L. Rothman

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Brain Energetics and Neuronal Activity

Brain Energetics and Neuronal Activity Applications to fMRI and Medicine

Editors R G Shulman and D L Rothman Yale University School of Medicine New Haven, Connecticut, USA

Copyright  2004

John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone (+44) 1243 779777

Email (for orders and customer service enquiries): [email protected] Visit our Home Page on www.wileyeurope.com or www.wiley.com All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England, or emailed to [email protected], or faxed to (+44) 1243 770620. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Other Wiley Editorial Ofﬁces John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA Wiley-VCH Verlag GmbH, Boschstr. 12, D-69469 Weinheim, Germany John Wiley & Sons Australia Ltd, 33 Park Road, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons Canada Ltd, 22 Worcester Road, Etobicoke, Ontario, Canada M9W 1L1 Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Library of Congress Cataloging-in-Publication Data Brain energetics and neuronal activity : applications to fMRI and medicine / editors, R. G. Shulman and D. L. Rothman. p. ; cm. Includes bibliographical references and index. ISBN 0-470-84720-4 (cloth : alk. paper) 1. Brain–Metabolism. 2. Energy metabolism. 3. Brain–Pathophysiology. 4. Brain–Magnetic resonance imaging. [DNLM: 1. Brain–metabolism. 2. Magnetic Resonance Imaging–methods. 3. Brain Diseases–physiopathology. 4. Energy Metabolism. 5. Neurons–metabolism. 6. Synaptic Transmission. WL 141 B8114 2004] I. Shulman, R. G. (Robert Gerson) II. Rothman, D. L. (Douglas L.) QP376 .B699 2004 612.8 2–dc22 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 0-470-84720-4 Typeset in 10/12pt Times by Laserwords Private Limited, Chennai, India Printed and bound in Great Britain by Antony Rowe Ltd, Chippenham, Wiltshire This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least two trees are planted for each one used for paper production.

Contents Contributors

vii

Foreword

ix

Section A: Background

1

1

Introduction Douglas L. Rothman and Robert G. Shulman

2

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States Louis Sokoloff

3

Techniques–MRS, fMRI, Robin de Graaf

4

Metabolic Modeling Analysis of Brain Metabolism Graeme Mason

13

C NMR, Indirect Detection of

3

13

C

11 31 53

Section B: Neuroenergetics and Activity

73

5

Cerebral Energetics and Neurotransmitter Fluxes Nicola R. Sibson

75

6

NMR Studies of the Metabolism and Energetics of GABA Neurotransmitter Pathways Kevin L. Behar and Douglas L. Rothman

99

7

Neural Energy Consumption and the Representation of Mental Events Simon B. Laughlin and David Attwell

8

Imaging Cerebral Metabolic Rate of Oxygen Consumption (CMRO2 ) using Approach at Ultrahigh Field Wei Chen, Xiao-Hong Zhu and Kamil Ugurbil

9

Deriving Changes in CMRO2 from Calibrated fMRI Fahmeed Hyder

147

10

Relationship between CMRO2 and Neuronal Activity Fahmeed Hyder and Hal Blumenfeld

173

111 17

O NMR

125

vi

Contents

Section C: Clinical Beginnings

195

11

NMR Studies of Bioenergetic Impairment in Human Epilepsy Hoby P. Hetherington, Jullie W. Pan and Dennis D. Spencer

197

12

MRS Studies of the Role of Altered Glutamate and GABA Neurotransmitter Metabolism in the 215 Pathophysiology of Epilepsy Ognen A. C. Petroff and Dennis D. Spencer

13

The Role of Altered Energetics of Neurotransmitter Systems in Psychiatric Disease Graeme F. Mason

239

Section D: Brain and Mind

257

14

Long-term Memory: Do Incremental Signals Reﬂect Engagement of Cognitive Processes? Jed A. Meltzer and R. Todd Constable

259

15

Using fMRI to Study the Mind and Brain Bruce E. Wexler

279

16

Brain and Mind: an NMR Perspective Robert G. Shulman and Fahmeed Hyder

295

17

The Role of the NMR Baseline Signal in the Study of Consciousness: the Restless Brain Robert G. Shulman

311

Index

315

Contributors David Attwell Department of Physiology University College London Gower Street London, WC1E 6BT, UK Kevin L. Behar Department of Psychiatry Yale University School of Medicine MR Center P.O. Box 208043 New Haven, CT 06520-8043, USA [email protected] Hal Blumenfeld Department of Neurology and Neurobiology Yale University School of Medicine P.O. Box 208018 New Haven, CT 06510, USA [email protected] Wei Chen Center for Magnetic Resonance Research Department of Radiology University of Minnesota Medical School 2021 6th Street SE Minneapolis, MN 55455, USA tel: 612-625-8814 [email protected] Robert Todd Constable Department of Diagnostic Radiology Yale University School of Medicine P.O. Box 208042 New Haven, CT 06520-8042, USA [email protected]

Robin de Graaf Department of Diagnostic Radiology Yale University School of Medicine MR Center P.O. Box 208043 New Haven, CT 06520-8043, USA [email protected] Susan Fitzpatrick James S. McDonnell Foundation 1034 South Brentwood Blvd, Suite 1850 St. Louis, MO 63117, USA [email protected] Hoby Hetherington Departments of Radiology, Physiology and Biophysics Albert Einstein College of Medicine Jack and Pearl Resnick Campus 1300 Morris Park Avenue Bronx, NY 10461, USA [email protected] Fahmeed Hyder Departments of Diagnostic Radiology and Biomedical Engineering Yale University School of Medicine MR Center P.O. Box 208043 New Haven, CT 06510, USA [email protected] Simon B. Laughlin Department of Zoology University of Cambridge Downing Street Cambridge, CB2 3EJ, UK [email protected]

viii

Contributors

Graeme Mason Department of Psychiatry Yale University School of Medicine MR Center P.O. Box 208043 New Haven, CT 06520-8043, USA [email protected]

Nicola Sibson Experimental Neuroimaging Group Department of Biochemistry University of Oxford South Parks Road Oxford, OX1 3QU, UK [email protected]

Jed A. Meltzer Department of Diagnostic Radiology Yale University School of Medicine MR Center P.O. Box 208042 New Haven, CT 06520-8042, USA

Louis Sokoloff National Institute of Mental Health National Institutes of Health Bethesda, MD 20892, USA [email protected]

Jullie W. Pan Departments of Neurology and Neuroscience Albert Einstein College of Medicine Jack and Pearl Resnick Campus 1300 Morris Park Avenue Bronx, NY 10461, USA [email protected] Ognen A.C. Petroff Yale University School of Medicine Department of Neurology P.O. Box 208018 New Haven, CT 06520-8018, USA [email protected] Douglas L. Rothman Department of Diagnostic Radiology Yale University School of Medicine MR Center P.O. Box 208043 New Haven, CT 06520-8043, USA [email protected] Robert G. Shulman Yale University School of Medicine MR Center P.O. Box 208043 New Haven, CT 06520-8043, USA [email protected]

Dennis D. Spencer Department of Neurosurgery Yale University School of Medicine 333 Cedar Street P.O. Box 208082 New Haven, CT 08520-8082, USA Kamil Ugurbil Center for Magnetic Resonance Research Department of Radiology University of Minnesota Medical School 2021 6th St. SE Minneapolis, MN 55455, USA Bruce E. Wexler Yale University School of Medicine Department of Psychiatry P.O. Box 208068 New Haven, CT 06520-8068, USA [email protected] Xiao-Hong Zhu Center for Magnetic Resonance Research Department of Radiology University of Minnesota Medical School 2021 6th St. SE Minneapolis, MN 55455, USA

Foreword My experiences over the past 30 years have led me to the realization that there are two kinds of researchers in the biological sciences: those who almost immediately recognize the centrality of metabolism to a full understanding of biological function and those who do not. The latter group always seems to represent a larger proportion of biological scientists, so that the focus of biological research tends to drift from studying metabolism into various phenomenological directions, until one realizes, as one inevitably does, that metabolism, particularly the metabolism of cellular energy production, what is often termed ‘intermediary metabolism’ is the key to inter-level analysis of biological systems. Metabolism provides the bridge from the genetic to the cellular and from the cellular to the organismic. Sooner or later, therefore, the hard search for truth comes back to addressing and unraveling the complexities of intermediary metabolism and abandoning the quest for a ‘magic bullet’. This return to a focus on metabolism is becoming unavoidable for scientists studying the inﬂuence of gene expression patterns on biological function. Likewise, as the chapters in this book document, cognitive neuroscientists searching for an understanding of the mechanisms by which neuronal assemblies process information can not avoid acquiring a deeper awareness of brain energy and intermediary metabolism. Brain structure – function relationships can only be clearly seen through a metabolic lens. As I neared completion of my doctoral research in the Laboratory of Cerebral Metabolism at Cornell University Medical College in 1984, I confronted the metabolism versus non-metabolism dichotomy head on. Although devoted to studying brain energy metabolism, my mentors advised me to forsake such studies and become more ‘molecular’ if I intended to have a viable research career. They had my best interests at heart; in their eyes, the era of traditional metabolic neurochemistry was on the wane. The classic approach of using metabolic encephalopathies (including ischemia, hypoxia, and hypoglycemia) to probe how perturbations in cerebral oxidative energy metabolism altered conscious states (e.g. lethargy, stupor, and coma) was becoming pass´e. To be fair, my advisors were not entirely incorrect. The general emphasis in neurochemical studies in the 1970s and 1980s concentrated on detailing how the supply of glucose and oxygen for energy metabolism supported neuronal function. The key observation (not yet made but described fully in this volume) needed to advance the ﬁeld was the critical link between the need to sustain the high rate of energy metabolism and its driving force – glutamate neurotransmitter turnover. Luckily, the solution to my career dilemma revealed itself while I browsed in the library. Serendipity, that often unacknowledged contributor to careers and discoveries, led me to one of the most important books I have ever read: Biological Applications of Magnetic Resonance edited by R.G. Shulman (1979 Academic Press New York, San Francisco and London). Shulman’s volume proﬁled several directions of research using nuclear magnetic resonance (NMR) spectroscopy to study metabolic reactions in vivo. It was immediately apparent to me that this approach, if applied to the brain, might be able to discover the Holy Grail of neurochemistry – the ability to monitor the metabolic correlates of neural information processing. In vivo NMR spectroscopy, because of its ability to monitor energy metabolism in the intact brain, in vivo, in real time, provided a novel approach for investigating the all-important tight coupling of cerebral metabolism and neuronal function. Not surprisingly, Bob Shulman’s laboratory at Yale University was

x

Foreword

already actively pursuing this line of research – and soon after provided me a new intellectual home. NMR spectroscopy breathed renewed life into the pursuit of what remains a somewhat elusive goal – unraveling the complex inter-relationships among cerebral function, cerebral structure, and cerebral metabolism. How closely the traditional tools of neurochemistry can monitor changes in the concentrations of intermediary and energy metabolites and accurately mirror real time in vivo levels is hampered by the unique dependence of brain function on blood ﬂow and cerebral metabolism as thoroughly documented in the chapter by Sokoloff. The few seconds of hypoxia, ischemia, or hypoglycemia inﬂicted during the time between obtaining brain tissue for study and doing the study unavoidably alters the concentrations of energy metabolites and destroys the functional cyto-architecture. During the 1970s a signiﬁcant percentage of neurochemists’ research efforts were directed toward developing ingenious methods to minimize experimental post-mortem changes of intermediary and energy metabolite concentrations. Researchers pursued every strategy to freeze the metabolite levels by limiting the effects of ischemia and hypoxia so as to mirror the in vivo concentrations present at the experimental time of interest Techniques such as in situ freezing preserved the structural integrity of the brain tissue, but risked uneven rates of inactivation of metabolic processes, particularly for deeper brain structures. The ‘brain blowing’ technique introduced by Bud Veech, a rather extreme apparatus using air pressure to extrude the brain from the cranium, provided shorter extraction and freezing times, but destroyed the anatomy. What was needed, if the ﬁeld was to progress, were methods that could monitor blood ﬂow and metabolism linked to normal cognitive and behavioral states in the intact brain, in vivo, in real time. The development of the 2-deoxyglucose (2-DG) autoradiography technique in the laboratory of Louis Sokoloff in the 1970s offered neurochemists the ability to measure regional rates of glucose utilization in animal models and to begin to link metabolism with physiology in the absence of induced pathology. Soon thereafter, positron emission tomography made possible, for the ﬁrst time, measurement of regional cerebral metabolic rates for glucose (FDG-PET) and oxygen (O-18 PET) in human subjects in real time as they engaged in behavioral tasks. For studies correlating regional changes in cerebral metabolism with regional changes in neural activity, 2-DG autoradiography and PET have some limitations. The analysis of 2-DG autobiographic data occurs in brain slices, post-mortem. Each individual animal in an experimental design provides data on cerebral metabolic rates for many different brain regions but for only a single time point. Human PET studies provide measurements of cerebral metabolism with excellent temporal series and spatial resolution, but the short-lived radio-isotope labeled compounds needed to conduct the experiments limited research opportunities. Furthermore, the need for intravenous injections of radio-isotopes limited the subject pool and essentially eliminated the possibility of pursuing certain types of studies, such as those involving healthy children. Additionally, 2-DG autoradiography and FDG-PET are not identical in every aspect, somewhat complicating comparisons between human and animal studies. In vivo NMR spectroscopy offered an alternative approach which allowed direct comparison of animal models with human subjects and was also not dependent on the use of radioactive tracers. As the development of in vivo NMR spectroscopic techniques for metabolic measurements was rapidly progressing, other applications of MR physics allowed scientists to obtain remarkably detailed anatomical images of the intact, living, human body. The brain images produced by magnetic resonance imaging (MRI) were particularly stunning; they revealed the 3-D complexity of the brain at a degree of resolution far surpassing that of contemporaneous imaging devices such as CAT scans. In the late 1980s, the physics of MRS and MRI led to the discovery of what could, arguably, be considered one of the 20th century’s most powerful advances in the ability to monitor brain activity in the intact subject. In 1990 Seiji Ogawa and colleagues at Bell Laboratories published the ﬁrst study using BOLD MRI, often referred to in the literature as functional magnetic resonance imaging or fMRI, to monitor ongoing brain activity related to ongoing behavior.

Foreword

xi

The ability to use PET and fMRI measurements to localize cognitive processes within a particular volume of neural tissue is based on the assumption that functional neuronal activity, and therefore metabolism, increase when a region is involved in the neural computations that implement a cognitive task. The functional neuronal activities involved in the communication of information between neurons include neurotransmitter synthesis, packaging, and release followed by the subsequent generation of action potentials. As explained above, the energy required for these and other brain processes is provided almost exclusively by oxidative glucose metabolism. Regions of increased functional energetic demand are identiﬁed by the corresponding increase in glucose metabolism, or the coupled parameters of the cerebral metabolic rate for oxygen (CMRO2) and the cerebral blood ﬂow rate (CBF). Depending on the radio-labeled tracer used, PET can measure glucose consumption, oxygen utilization, or CBF. Interpreting the fMRI BOLD signal changes is somewhat more complicated because BOLD is sensitive to both CMRO2 and CBF. The cognitive neuroscience community quickly embraced BOLD fMRI despite the pitfalls of equating changes in a signal comprising several complex physiological parameters to changes in neuronal ‘activity’. Cognitive neuroscience emerged as a unique academic discipline in the late 1980s from the deliberate efforts of scientists interested in identifying the neural correlates of cognition by integrating the experimental and theoretical traditions of cognitive psychology, systems and computational neuroscience, linguistics, and philosophy. BOLD fMRI seemed ideally suited for pursuing cognitive neuroscience’s stated goals. Cognitive neuroscientists’ enthusiasm for fMRI studies led to a rapid and exponential growth in institutional MR centers. These centers concentrated their resources on developing methods for marrying psychological studies to functional imaging. Unfortunately, their lack of interest in pursuing metabolic or mechanistic research meant there was not a corresponding commitment to nurturing brain research with in vivo NMR spectroscopy. The lack of an appreciation by many cognitive neuroscientists (particularly those entering the ﬁeld primarily trained as psychologists or systems neuroscientists) of the centrality of energetics to understanding how information is processed in the brain has resulted in a cognitive neuroscience that acts as though there has not been a century of effort expended on unraveling the mechanisms coupling metabolism, blood ﬂow, and function in the brain. Cognitive neuroscientists interpreting changes in the BOLD signal as direct measurements of the neural correlates of human cognition and behavior without understanding the energetic and metabolic correlates of neural activity stand on theoretically shaky ground. It is the energetic and metabolic correlates of neuronal information processing that are coupled to the hemodynamic signals. There are several important questions integral to the understanding of the neural basis of cognition that cannot be answered with functional imaging studies but that can be investigated with MR spectroscopy. Functional neuroimaging studies proceed from an assumption that the observed signal changes reﬂect neuronal activity, even though other cell types, most notably astrocytes, are known to be important contributors to overall brain energy metabolism and neurotransmitter function. Pierre Magistretti and his colleagues have published a series of papers identifying the astrocyte as the primary site of cerebral glucose consumption. Magistretti has proposed that astrocytic glucose uptake is mechanistically coupled to neuronal energy requirements via non-oxidative glycolysis to provide energy for removing glutamate, an excitatory neurotransmitter released by the nerve terminals, from the synaptic cleft. The lactate produced as a result of the incomplete oxidation of glucose is then transported from the astrocyte to the neuron as a substrate for oxidative energy metabolism. The chapter by Sibson summarizes the in vivo MRS results from rat and human brain detailing the ﬂux from glucose to glutamate that support and extend Magistretti’s ﬁndings. 13 C NMR spectroscopic studies have shown that the rate of astrocytic glutamate uptake increases with glucose metabolism at a close to 1:1 stoichiometry. In vivo MRS ﬁndings offer insights into the underlying nature of the BOLD signal. This statement does not imply that MRS research should be embraced merely because it offers the hope of ‘quantitating’ BOLD. Rather, as is made clear by several chapters in this volume, MRS functional neuro-energetic studies offer

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Foreword

cognitive neuroscientists the ability to pursue an entirely new level of analysis of brain function. The series of PNAS papers by Hyder et al., described in detail in this volume, provides a biological underpinning for theoretical models of unconscious and conscious information process such as that proposed recently by Stan Dehaene and Jean Pierre Changeux. Slowly but surely, cognitive neuroscience is becoming aware that the small physiological differences detected by BOLD imaging are the mere tip of an iceberg. Functionally, the high rate of ongoing neuronal activity, representing close to 80 % of total cerebral energy metabolism, may be of greater interest. In vivo MRS studies, together with fMRI, allow us to build an integrated science of brain/behavior by bridging from cognition to cerebral oxidative energy metabolism and by linking neuronal information processing with glutamate neurotransmitter function. It is rare to discover a single volume that both summarizes and celebrates decades of biological research while also pointing out a path to the future. In 20 years I have come across only two. Not surprisingly, they both emphasize that we cannot understand biological function without understanding metabolism and both are edited by Bob Shulman. Susan Fitzpatrick September 2003 http://anestit.unipa.it/reference/bibliography/snacc/Metab3.html

Section A Background

1 Introduction Douglas L. Rothman and Robert G. Shulman Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

1.1 1.2 1.3 1.4

Background Energy and Neurotransmission Clinical Beginnings Brain and Mind

5 6 7 8

This book illustrates and celebrates recent NMR investigations of brain, identifying its molecular constituents and following their metabolic activities in vivo. This physical, spectroscopic method is anchored in its two origins – its present technical methods descended from Bloch and his colleagues and its present metabolic information derivable from the simple, beautiful NMR experiments on water of Bloembergen, Purcell and Pound. Water continues as an endlessly fascinating material and in the present volume plays the central role in MRI and fMRI experiments. From the beginning NMR studies have depended upon interaction between atomic nuclei and their chemical environment and have been valuable reporters of these environments. These interactions have been readily interpreted because of two simplifying characteristics of the NMR phenomena. First spin 12 nuclei of the common elements, e.g. hydrogen, carbon and phosphorus, interact very weakly with their environment relative to the strong forces they can experience in available laboratory magnetic ﬁelds. Accordingly environmental interactions affecting chemical shifts, multiplet structure and relaxation times, being weak compared to these Zeman interactions, are perturbations and as such are readily and quantitatively evaluated. The second simplifying property that allows reliable information about the nuclei’s surroundings is that NMR phenomena, in contrast to most spectroscopic methods, take place in the ground electronic state, and thereby provide a well-established basis for interpreting the perturbations by the molecular environment. As a quantitatively understandable measure of molecules and their interactions NMR has grown continually in these years. It has become a prime method Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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Brain Energetics and Neuronal Activity

for the study of matter – in chemistry, condensed matter physics, geology, biochemistry and in all studies of materials. Studies of brain metabolism are playing a growing role in neuroscience. Cerebral metabolic pathways date back to the exciting beginnings of physiological chemistry, when Charles Sherrington proposed that ‘the blood supply of any part of the cerebral tissue is varied in accordance with the activity of the chemical changes which underlie the functional activation’. Activities of brain were then to be explained by the same criterion that physiologists were using in exploring the chemical basis of muscle and heart. Methods for measuring cerebral inputs of glucose and oxygen that have been developed since 1950 have established the basic dependence of cerebral energetics upon the oxidation of glucose. Cerebral blood ﬂow has been measured by similar methods so that neurophysiology has shown that blood ﬂow follows energy consumption and thereby links brain chemistry with the organism. In more recent years non-invasive methods of nuclear magnetic resonance and positron emission tomography (PET) have played increasingly important roles in brain studies. Magnetic resonance imaging (MRI) has revealed cerebral anatomy with great speciﬁcity and has helped to localize the more chemical ﬁndings of PET, functional magnetic resonance imaging (fMRI), and magnetic resonance spectroscopy (MRS). These metabolic methods have now been developed to the point that their ﬁndings contribute substantially to brain science. This book describes recent contributions of magnetic resonance methods to studies of brain energetics and function. The value of magnetic resonance, particularly MRI, has been evident from its ﬁrst human applications. High-resolution non-invasive images moved clinical studies into a better-informed era. A little more than a decade after, fMRI made parallel advances in the localization of stimulated brain activity. MRS, particularly in measurements by 13 C-label ﬂow, has provided information relating brain metabolism and neurotransmitter ﬂows. The contributions of 13 C MRS to brain studies form the unifying theme underlying this book. The insights into metabolism, provided by MRS and PET, have depended to some extent upon the great powers of MRI and fMRI while, in turn, they have led to some re-interpretation of these imaging methods. Together these magnetic resonance methods have opened new understandings of brain function and now offer new directions for research. Although in retrospect a smooth research path can be traced through the past decades leading to a metabolic model of brain function, nonetheless certain obstacles to research planning unique to the brain had to be overcome. The very nature of the work done by the brain was not as obvious as in other organs. Muscle work was readily deﬁned and measured as force through a distance. Nor did muscle power expenditures, easily measured as the rate of work, present any conceptual difﬁculties. The chemical basis of cerebral work, like the analogous work done by the heart, is now being expressed by the studies presented in this book, in similar terms, as molecules and ions pumped across gradients by ATP. We have obtained this analogous deﬁnition and quantitation of brainwork from the experimental (Sibson) and theoretical (Laughlin) research described in Chapters 5 and 7. However, these very recent results had to overcome paradigmatic ideas about the unimportance of energy for brainwork that had been formed on the basis of very few studies. Until recently the generally accepted belief in the ﬁeld, from a comparison with the squid axon, considered only ∼1 % of brain energy production to be devoted to neuronal ﬁring. Only when the multi-synapses of the mammalian brain were speciﬁcally included did calculations support the MRS results that nearly all the brain energy was devoted to neuronal ﬁring. The negligible role assumed for cerebral energetics had been reinforced by the suggestion that stimulated brain activity was supported by non-oxidative glycolysis despite the main energy source being the oxidation of glucose. The recent stoichiometries showing efﬁcient use of glucose oxidation for energy had to overcome these widespread views. Perhaps the more strongly held obstacles came from residual phenomenological dualism. It was difﬁcult to realize that the thinking process of mind required work, based upon the hydrolysis of ATP, just as

Introduction

5

a muscle did work when lifting weights. This traditional difﬁculty in formulating brainwork slowed the efforts to calculate such work energetically. But that relationship has now been quantiﬁed as described in the following pages, and provides a sturdy foundation for the study of the more complex aspects of brain. As this understanding advances it leads to consideration of the interactions between these aspects of neuroscience with the broad ﬁeld of psychology. To what extent does modern psychology further our understanding of neurophysiology or, conversely, in what way does the understanding gained about brain activity and energetics advance psychological conceptualization? Functional imaging of brain localizes brain activity non-invasively and quickly – but how does this methodology illuminate our understanding of brain activity? And how does understanding energy consumption help to expand the scope of functional imaging? Finally, underlying all these questions, how does brain energy metabolism relates to neuroscience, the study of brain components, structure and action at the cellular and molecular level? Out of the broad ﬁelds that are presently actively studying the functioning brain, the studies reviewed here have focused upon a simple concept, namely brain energy consumption and neuronal activity are chemical processes. Brain activity is not merely powered by chemical reactions – that activity, in fact, consists of chemical reactions. Brain energetics compares the production and use of energy, usually mediated through ATP, in which glucose is metabolized. Neuronal activity is the sum of chemical reactions by which information is processed. This activity we show can be proﬁtably followed by measurements, in vivo, of the rates of chemical reactions. Chemical reactions in vivo do not occur in isolation, as they do in a test tube. In vivo all reactions are linked to others and the linked reactions form a network that is conveniently deﬁned as metabolism. In the brain the chemical reactions of ion ﬂuxes across membrane, neurotransmitter processing and the electrical actions of neuronal spiking are all coupled to energy consumption and the control of metabolism. The unique perspective of this book derives from the ability to measure cerebral metabolic ﬂuxes, in the living human and animal brains, by in vivo NMR methods. These quantitative assessments of metabolite concentrations and rates of reaction, in vivo, provide the unique, strong data set, that has allowed metabolism, studied this way, to provide a platform for novel insights into biological function.

1.1. BACKGROUND The ﬁrst four chapters provide background information of a historical and technical nature. The NMR studies of the brain, which form the core of this book are discussed in the early chapters. NMR methods, magnetic resonance spectroscopy (MRS) have measured in vivo concentrations and kinetics of molecular metabolites. These small molecules, e.g. glutamate, glutamine, lactate, glucose, etc., are studied as the components of energetic and neurotransmitter pathways. The early physiological studies of cerebral glucose and oxygen uptake established the chemical basis of brain energetics, showing that glucose oxidation is the main energy source, have been summarized by Lou Sokoloff, a pioneer of this ﬁeld, in Chapter 2. Dr. Sokoloff’s development of the deoxyglucose tracer extended these studies, which originally were primarily based on arterial venous difference measurements, to high resolution mapping of brain functional activity based upon glucose uptake and ﬂow. This deoxyglucose mapping remains the gold standard to which more recent non-invasive modalities such as fMRI are compared. In addition these studies also formed the basis for the development of PET studies of human glucose metabolism using the deoxyglucose tracer. A key ﬁnding in these studies, the understanding that glucose oxidation provides the brain energy, was re-inforced and conﬁrmed in vivo by 13 C MRS measurements of the ﬂux from labeled substrates particularly 1-13 C glucose, through the (tricarboxylic acid) TCA cycle. Numerical values of the cerebral metabolic rate of oxygen consumption obtained from these MRS measures of TCA cycle ﬂux agreed with the results from the deoxyglucose autoradiography and PET methods in animals and humans respectively. Chapter 3 by Robin de Graaf introduces NMR methods used in the book. NMR of cerebral metabolism obtains quantitative measurements by combining the regional speciﬁcity of imaging methods with the

6

Brain Energetics and Neuronal Activity

chemical identiﬁcation and quantitation of metabolites which has been developed extensively by high resolution NMR. The description is at a simple level, suitable for anyone who has taken (and remembers some of) a course in organic chemistry. From this base more advanced methods used in MRS studies are developed and are referred to throughout the other reports. Since the core of quantitation lies in hypothesizing and evaluating a model of in vivo ﬂuxes, this subject is reviewed in Chapter 4 by Graeme Mason. ‘Modeling’ of data is used in a very positive way in these studies, in that the ‘model’ represents bundled hypotheses about the in vivo reaction pathways. As such the models serve to evaluate ﬂuxes from data but also serve to test the hypothesis about the parameter being evaluated. In a particularly simple application, a model predicts that the value of a particular ﬂux must be the same (within experimental error) when that ﬂux is measured in different, independent experiments. Values of rates obtained by modeling the data depend upon statistical ﬁts of the model to the data. A simpliﬁed, yet informative description of such calculation forms a central feature of this chapter.

1.2. ENERGY AND NEUROTRANSMISSION Section B includes descriptions of the experimental and theoretical determinations of in vivo metabolic rates, energy consumption in the form of oxygen usage and neuronal work done by neurotransmission and spike activity. A key novel ﬁnding of this work is that energy consumption is coupled to the release of the neurotransmitter glutamate upon neuronal ﬁring and its subsequent recycling through glutamine. This ﬂux was proposed in the 1970s and was established as the major pathway by the 13 C experiments. In Chapter 5 by Nicola Sibson, studies are described in which 13 C-labeled glucose and other labeled precursors are followed into glutamate and glutamine pools in human and animal brains by in vivo 13 C MRS. The rate of label appearance in glutamate has provided a measure of the tricarboxylic acid cycle ﬂux VTCA , which is a direct measure of neuronal energy consumption. The ﬂux into the glutamine pool, measured in the same experiment by in vivo 13 C MRS, is used to quantitate the rate of glutamate neurotransmitter release and recycling. The quantitation shows that there is a one-to-one change of glutamate neurotransmitter ﬂux with neuronal glucose oxidation, i.e. for each additional molecule of glutamate released as a neurotransmitter there is one molecule of glucose oxidized in neurons. This stoichiometry allows changes in the cerebral rate of energy consumption to be converted directly into change of neuronal signaling, and provides key information for understanding the energetic and molecular mechanisms which couple glutamate neurotransmission to energy consumption. The proposal linking the in vivo results to the cellular ﬁndings of Magistretti and coworkers that astroglial glutamate uptake depends on glycolytic ATP are described. It provides a chemical measure of neuronal activity, which has previously been undeﬁned, and in conformity with the message of this book, shows that the rate of neuronal activity is deﬁned by the ﬂux through a metabolic pathway. These total ﬂuxes of glutamate to glutamine neurotransmitter cycling reﬂect the sum of glutamate and GABA neurotransmitter ﬂow. GABA is the most abundant inhibitory neurotransmitter in the cortex. Chapter 6, by Kevin Behar and Douglas Rothman, shows how GABA neurotransmitter release and cycling can be distinguished from the total glutamate to glutamine cycling. The contributions of GABA to the total glutamate plus GABA neurotransmitter cycling ﬂux has been shown to be signiﬁcant. The chapter describes how the use of other labeled substrates, in particular labeled acetate, allows this distinction to be made, and sets the stage for future studies to understand the relationship between inhibitory and excitatory neurotransmission and energetics, as well as subcellular metabolic compartmentation of GABA synthesis. In Chapter 7, Laughlan and Attwell review their energy budget for glutamate neurotransmitter cycling. Taking literature values for the different steps in the pathway of neuronal ﬁring with glutamate release and cycling they have broken the energy requirements into different steps. Most of this energy is consumed in pumping the neuronal ion gradients after ﬁring with approximately equal energies devoted to pre- and postsynaptic pumping. As expected the energy requirements for the chemical steps of glutamate release and

Introduction

7

conversion to glutamine and recycling back into the glutamate vesicles is small compared to the neuronal energy requirements. Assuming the glutamate concentration of vesicles, the efﬁciency of neuronal ﬁring and based on a ﬁring rate of 4 Hz, they ﬁnd reasonable agreement with the observation by 13 C MRS that a large majority of the energy is devoted to neuronal activity and only ∼20 % is devoted to non-functional neuronal energy needs commonly called housekeeping. Their energy budget provides a valuable framework for relating energy consumption, measured by 13 C MRS methods to detailed energetic sub-processes. It also provides a framework for studies combining MRS and MRI with inhibitors of speciﬁc energetic sub-processes to test the understanding of the energetic basis of function in detail. The next two chapters present very recent NMR methods for measuring the basic neuroenergetic parameter, CMRO2 . High magnetic ﬁelds have increased NMR sensitivity so that direct 17 O NMR experiments can determine the rate of oxygen consumption by following the label ﬂow to H17 2 O, as shown in Chapter 8 by Wei Chen. These quite direct measurements of oxygen usage are a substantial advance in sensitivity over previous 1 HNMR methods using indirect detection of H17 2 O relaxation times. Furthermore they have potentially similar spatial and temporal resolution as PET CMRO2 measurements, with the advantages of more accurate quantitation and the ability to perform multiple repeated studies due to the absence of radioactive isotope. Careful studies of this direct measurement of CMRO2 have elucidated the methodology of similar PET measurements. The BOLD fMRI, is known to depend upon complex interactions of NMR relaxation times, CMRO2 , CBF and Cerebral Blood Volume (CBV). In Chapter 9, Hyder describes his method of determining CMRO2 from BOLD measurements. This required associated measurements of CBF by a spin tagging imaging experiment, of CBV by the use of iron contrast reagents and of the spin-spin relaxation times. Values of CMRO2 agreed with values obtained by an indirect 1 HNMR detection of the 13 C turnover of glutamate. The high resolution results of this method, giving increments of CMRO2 can, by connections to absolute CBF – spin tagging results, be converted to absolute values. Values of CMRO2 obtained this way are compared with the values measured by the ﬂows of 13 C-labeled glucose in the same experiments. This calibration brings together functional imaging and metabolic energetics. In place of the phenomenological connections offered by other investigators between functional imaging signals and arbitrarily selected variables, this calibration relates the fMRI signal mechanistically to a meaningful neurophysiological parameter. Once CMRO2 is derived from the BOLD signal in this way, the relationships between energy and neuronal activity established throughout the earlier chapters become available with the temporal and spatial resolutions of the calibrated BOLD measurement. This connection forms the basis of interpreting the BOLD effect in later chapters. Both of these newer methods of determining CMRO2 are shown to agree with classical methods of oxygen consumption with signiﬁcant advantages over earlier methods. In Chapter 10 single electrode recordings of neuronal ﬁring rates are regionally coordinated with values of CMRO2 obtained from calibrated BOLD measurements. Neuronal ﬁring rates, or spiking, are for many the ‘gold standard’ of neuronal activity. To measure these rates, individual neurons are identiﬁed in the rat somatosensory cortex, located during forepaw stimulation by BOLD experiments, and their ﬁring rates followed through changes in depth of anesthesia and during sensory stimulation. The ensemble of neurons, followed in this way, correlates deployments of the neuronal population with the total energetics. Changes during the stimulation are shown to involve the majority of neurons in the voxels, rather than a selected few. Implications of these results for neuronal function are explored here, and in several of the subsequent chapters.

1.3. CLINICAL BEGINNINGS Applications of magnetic resonance results to epilepsy and the psychiatric diseases clearly indicate directions for future study. Mesial temporal lobe epilepsy, one of the most common and debilitating forms, has

8

Brain Energetics and Neuronal Activity

been shown by these measurements in conjunction with PET and microdialysis to be a disease in which the energetics and the glutamate neurotransmitter cycling have been impaired. In Chapter 12, Petroff and Spencer describe applications of 13 C MRS to the glutamate glutamine cycling in the hippocampus. They show that the degree of neuronal loss/damage in the hippocampus correlates well with the degree of impairment. A model is proposed in which astroglial metabolic impairments lead to reduced glutamate uptake, explaining the elevated extracellular glutamate (and resultant hyperexcitability) during even the quiescent interictal periods between seizures. The concentration of NAA is shown by Hetherington et al. in Chapter 11 to depend upon the energetic state of the mitochondria NAA decreases, reﬂecting a decrease in mitochondrial energetics, are proposed to be accompanied by more rapid release of glutamate. The ﬂux from neurons is measurable by 13 C MRS. Combining the results from this two pronged study of temporal lobe epilepsy we see that (1) the epileptic focus has lower NAA; (2) this reﬂects decreased mitochondrial energetics which (3) leads to a more rapid release of glutamate, thereby suggesting a cause for the (4) high concentrations of extracellular glutamate previously observed. An additional mechanism that presumably contributes to the high glutamate levels is seen in (5) the decreased rate of glutamate neurotransmitter cycling in the epileptogenic focus. Clearly these novel, sturdy in vivo insights offer interventional opportunities for preventing epileptic seizures. From another direction the concentration of GABA has been shown to correlate quantitatively with the mood of normal subjects. The variation of GABA levels during alcoholism and in the subsequent withdrawal period has promise for understanding features of addiction. In the more daunting problems of depression and schizophrenia, preliminary results of neurotransmitter activity give promising correlations. The approach from magnetic resonance as from so many directions is mainly conﬁned to phenomenological correlations. The problem faced by magnetic resonance in studying mental illness resembles that of other physical methods, immersed as they all are in the cyclical problem of having to deﬁne the problem while studying it. The deﬁnite information about the depressed condition reported by Graeme Mason in Chapter 11 provides an exciting starting point for future studies.

1.4. BRAIN AND MIND Several implications of the metabolic studies for the planning and interpretations of fMRI experiments are proposed in the book. The interpretations of fMRI experiments in light of these results are discussed in this section. The metabolic results are presented in six chapters of Section B and interpreted in Chapter 16. They emphasize that the differencing methods used in fMRI experiments, measure changes in neuronal activity between the baseline and stimulated conditions. fMRI by discarding the baseline activity neglects the majority of neuronal activity which has been measured in Chapters 5 and 10 where it was shown to participate in the brain’s response to the stimulus. One implication for the usual functional imaging signal is that, while increases or decreases of signal show activity changes in the region, still the absence of changes does not mean the absence of activity. Furthermore the magnitude of the change, being dependent upon the baseline activity, does not measure the magnitude of brain activity during the task. Chapter 14 applies some of these concepts to survey and evaluate fMRI studies of declarative memory in humans. The conventional differencing method has been employed in the experiments surveyed and information about the pathways of memories reviewed to show how differencing, if used carefully, can illuminate mental processes. It is suggested that the absence of incremental signals in the hippocampus during traditional memory tasks arises from the high baseline activities, in accordance with the 13 C MRS results. The wide variety of enriched memory tasks that can provoke incremental fMRI signals are examined closely in the search of a unifying conceptual basis. Chapter 15 reviews selective fMRI results to explore the accomplishments and limitations of localizing activations. Beyond the static mapping of brain responses this chapter investigates effects of the changing

Introduction

9

nature of the response, of the task and of the subject upon the meaningfulness of the results. Chapter 16 considers the consequences of the present metabolic ﬁndings for the investigation of complex brain activities including subjectivity. It emphasizes how the quantitative description of baseline neuronal activity, reached in the early chapters, should not be ignored by any psychology of mind. In the resting awake state, there is a high level of cortical energy consumption dedicated to neuronal signaling. It is suggested that the high baseline activity is caused by wide neuronal connections which support consciousness. These results describe a brain that accommodates and is intrinsically dependent upon subjective experience. The metabolic ﬁndings of highly active baseline activities are proposed to provide boundary conditions for a view of mind in which subjectivity and consciousness are not neglected. The last chapter summarizes conclusions reached in the book, and suggests possible future directions.

2 Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States Louis Sokoloff Laboratory of Cerebral Metabolism, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA

2.1 Introduction 2.2 Local Cerebral energy Metabolism 2.3 Biochemical Mechanisms of Functional Activation of Energy Metabolism at the Tissue Level 2.4 Localization of Functionally Activated Energy Metabolism at the Tissue Level 2.5 Cellular Localization of Functional Activation of Energy Metabolism 2.5.1 Effects of Increased Extracellular K+ and Intracellular Na+ Concentrations 2.5.2 Effects of Glutamate on Glucose Utilization in Astroglia 2.6 Summary

11 13 17 19 24 24 25 27

2.1. INTRODUCTION The biochemical pathways of energy metabolism in brain are in most respects like those of other tissues, but special conditions peculiar to the central nervous system produce obstacles to studies of intermediary metabolism of the brain in vivo. For example, the blood–brain barrier restricts the exchange of substrates and products between brain and blood, and, consequently, biochemical reactions normally operating within the brain tissues are often not detectable with traditional in vivo methods based on exchange of substrates and metabolites between the blood and brain. In vitro studies best serve to identify pathways of intermediary Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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Brain Energetics and Neuronal Activity

metabolism, biochemical mechanisms, and potential rather than actual performance, but valid identiﬁcation of the ultimate substrates and products of cerebral energy metabolism and their rates of utilization and production under conditions of normal brain function can be obtained only in vivo. The ﬁrst deﬁnitive studies of cerebral energy metabolism in vivo began with the development of the nitrous oxide method by Kety and Schmidt (1948). This method made it possible to determine the rates of cerebral blood ﬂow (CBF) and oxygen consumption (CMRO2 ). Originally designed for use in man it was also applicable in animals, and, furthermore, it could be used in the unanesthetized state, a particularly important requirement to study the normal functioning brain. The method was based on the indirect Fick principle which states that, when a chemically inert tracer (i.e. one that is neither consumed nor produced by a given tissue) is introduced into the circulation, the rate of change in its content in any tissue is equal to the difference between the rates at which it is brought to that tissue in the arterial blood and removed from it in the venous blood. The method originally employed low concentrations of nitrous oxide as the tracer but was subsequently adapted for use with inert radioactive gases, e.g., 85 Kr, 79 Kr, and 133 Xe. Inasmuch as application of the Fick principle requires sampling of both arterial and representative cerebral venous blood for determination of their tracer concentrations, cerebral arteriovenous differences for a variety of substrates and products of cerebral metabolism could also be measured and their rates of utilization or production by the brain calculated as the product of these arteriovenous differences and CBF. Numerous studies with this method established that cerebral energy metabolism is extraordinarily active in the unanesthetized state (Clarke and Sokoloff, 1999). For example, in normal, conscious, young adult men the brain comprises only about 2 % of total body weight, but it alone consumes about 20 % of the total resting body oxygen consumption. Studies with the method also established that glucose is not only an essential but normally the almost exclusive substrate for the brain’s energy metabolism. Under normal conditions the brain utilizes glucose in almost stoichiometric amounts with the oxygen required for the complete oxidation of glucose to CO2 and H2 O (Table 2.1). For complete stoichiometry 6 mol of O2 are consumed and 6 mol of CO2 produced per mole of glucose utilized, i.e., a molar ratio (O2 /glucose ratio) of 6.0; the respiratory quotient (R.Q.) (ratio of CO2 production to O2 consumption) is then 1.0. Under normal conditions cerebral glucose utilization (CMRglc ) not only accounts for all the O2 consumed, but exceeds the rate for complete stoichiometry with CMRO2 by about 20 %, and the measured O2 /glucose ratio is about 5.5 (Table 2.1). In ketotic states, such as those due to starvation, fat-feeding, diabetes, etc., the blood levels of the ketone bodies, D-β-hydroxybutyrate and acetoacetate, are elevated, and these can substitute partly but not completely for glucose as the substrate for the brain’s Table 2.1. Rates of and relationship between cerebral oxygen consumption and glucose utilization in normal, conscious, young adult men Function

Rate

O2 consumption (µmol/100 g of tissue/min) Glucose utilization (mmol/100 g of tissue/min) O2 /glucose ratio (mol/mol) Glucose equivalent of O2 consumption (µmol/100 g tissue/min) (assuming 6 mol O2 per mol of glucose) Excess of total glucose utilized above glucose oxidized (mmol/100 g of tissue/min) CO2 production (µmol/100 g of tissue/min) Cerebral respiratory quotient (R.Q.)

156 31 5.5 26 5 156 0.97

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States

13

oxidative metabolism (Owen et al., 1967; Krebs et al., 1971); in such cases the O2 /glucose ratio rises above 6.0. Numerous studies with the nitrous oxide method and its derivatives demonstrated that cerebral energy metabolism declines in parallel with reduced levels of consciousness. For example, CMRO2 was moderately lower in conditions in which the sensorium was impaired but not to the level of unconsciousness, e.g., diabetic acidosis, moderate hypoglycemia, cerebral arteriosclerosis, etc., and markedly depressed in conditions with complete unconsciousness or coma, e.g., general anesthesia, diabetic coma, insulin coma, hepatic coma, and cerebral ischemia due to increased intracranial pressure caused by brain tumors (Kety, 1950; Clarke and Sokoloff, 1999). All these were, however, pathological conditions in which energy metabolism was probably reduced due to disrupted intracellular biochemical processes, insufﬁcient substrate supply, or lesser demand for energy because of pathologically depressed neuronal functional activity. In physiological and pharmacological states with obviously altered mental functions, not necessarily altered levels of consciousness, such as in schizophrenia, LSD intoxication, mild alcoholic inebriation, sedation, mental exercise during solving of arithmetic problems, etc., no changes in CMRO2 were found. The failure of the nitrous oxide method to detect a relationship between energy metabolic rate and functional activity in the brain was undoubtedly due to the fact that it measured only average rates of oxygen and glucose utilization in the brain as a whole and not in the speciﬁc regions of the brain where the functional activities were localized.

2.2. LOCAL CEREBRAL ENERGY METABOLISM The capability to determine local rates of energy metabolism in speciﬁc regions of the brain in vivo ﬁrst became available with development of the 2-[14 C]deoxyglucose ([14 C]DG) method (Sokoloff et al., 1977). This method measures local rates of glucose utilization simultaneously in all regions of the nervous system. Furthermore, it is fully applicable to unanesthetized animals, thus avoiding the complications of alterations of neural functions and metabolism by anesthetic agents. Because of the close to stoichiometric relationship between oxygen and glucose utilization in brain, glucose utilization is generally as good a measure of cerebral energy metabolism as oxygen consumption. 2-[14 C]Deoxyglucose (2-[14 C]DG) was used because it is an analog of glucose with which it competes for blood–brain transport into the brain and for phosphorylation by hexokinase to their hexose-6-phosphate derivatives. Unlike glucose-6-phosphate, however, which is very rapidly metabolized further via the glycolytic and tricarboxylic pathways ultimately to H2 O and CO2 , 2-[14 C]DG-6-phosphate can be metabolized to some extent to 2-[14 C]DG-1-phosphate, 2-[14 C]DG-1-6-phosphate, and 14 C-labeled glycogen, but all the products of 2-[14 C]DG phosphorylation remain trapped in the cells. Consequently, all the labeled products of 2-[14 C]DG phosphorylation accumulate with time and are retained within the cells with negligible loss for considerable lengths of time. From a kinetic analysis of the behavior of glucose and tracer concentrations of 2-[14 C]DG in brain an operational equation was derived with which to compute the local rates of CMRglc from the measured times courses of arterial plasma glucose and 2-[14 C]DG concentrations and the local tissue isotope concentration at the end of the experimental period, usually 30–45 min. Local 14 C concentrations in identiﬁable anatomical structures throughout the brain are determined by the use of quantitative autoradiography (Figure 2.1), a technique originally developed for measurement of local cerebral blood ﬂow in animals with chemically inert, diffusible radioactive tracers (Landau et al., 1955; Freygang and Sokoloff, 1958). The autoradiographs obtained with the 2-[14 C]DG method can be converted by computerized image-processing techniques into color-coded quantitative images of the brain in which local rates of CMRglc are encoded in the color scale. These processed autoradiographic images provide, therefore, the means to visualize local rates of glucose utilization throughout the brain exactly where they occur with a spatial resolution of 100–200 µm (Smith, 1983a).

14

Brain Energetics and Neuronal Activity

Autoradiographic calibration 1200

Standard concentration (nCi/g)

Tissue concentration (nCi/g)

1000 Best-fit standard concentration (nCi/g) 800

600

400

200

0 0

0.2

0.4

0.6 0.8 Optical density

1

1.2

1.4

Figure 2.1. [14 C]DG autoradiographs of conscious rat brain and of calibrated [14 C]methylmethacrylate standards as well as the calibration curve between optical density and tissue 14 C concentrations derived from densitometry of the standards.

Autoradiography is, of course, not applicable to man. To adapt the [14 C]DG method for use in man it was necessary to introduce a γ -emitting radioactive label into 2-deoxyglucose to allow external detection of the tracer. This was done by attaching 18 F to the carbon in the 2-carbon position of 2-deoxyglucose to produce 2-[18 F]ﬂuoro-2-deoxy-D-glucose (18 FDG), and the local tissue concentrations of label were determined by external scintillation counting with a single photon emission scanner in place of autoradiography (Reivich

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States

15

et al., 1979). 18 F is a positron emitter, and the 18 FDG method was subsequently adapted further for use with positron emission tomography (PET) (Phelps et al., 1979). PET provides better accuracy and spatial resolution than single photon detection although its spatial resolution in the mm range is still at least an order of magnitude below the 100–200 µm of the autoradiographic [14 C]DG method. Applications of the [14 C]DG and 18 FDG methods in both animals and man showed that CMRglc varies widely in the various structures of the brain. It is much higher in gray matter than in white matter and is generally reduced by anesthesia (Table 2.2) (Sokoloff et al., 1977). It was also clearly established that neuronal functional activation stimulates energy metabolism in neural tissues just as it does in other tissues (Sokoloff, 1981). The magnitude of the increases in glucose utilization is quantitatively related to the intensity of functional activation. For example, retinal stimulation by randomly timed light ﬂashes raises local CMRglc in proportion to the logarithm of the light intensity in those structures of the brain that receive direct projections from the retina (Figure 2.2). (Miyaoka et al., 1979; Sokoloff, 1981). Electrical stimulation of the cervical sympathetic trunk or sciatic nerve increases CMRglc linearly with spike frequency in the Table 2.2. Local rates of cerebral glucose utilization in rats in the conscious state and under light thiopental anesthesia (means ± SEM) Structure

Conscious (6)

Anesthetized (8)

% Effect

Gray Matter 111 ± 5 157 ± 5 107 ± 3 118 ± 3

64 ± 3 81 ± 3 65 ± 2 67 ± 2

−42 −48 −39 −43

Lateral Geniculate Nucleus Medial Geniculate Nucleus Thalamus: Lateral Nucleus Thalamus: Ventral Nucleus Hypothalamus Caudate-Putamen Hippocampus: Ammon’s Horn Amygdala

92 ± 2 126 ± 6 108 ± 3 98 ± 3 63 ± 3 111 ± 4 79 ± 1 56 ± 4

53 ± 3 63 ± 3 58 ± 2 55 ± 1 43 ± 2 72 ± 3 56 ± 1 41 ± 2

−42 −50 −46 −44 −32 −35 −29 −27

Cochlear Nucleus Nuclei of Lateral Lemniscus Inferior Colliculi Superior Colliculi Superior Olivary Nucleus Vestibular Nucleus Pontine Gray Matter Cerebellar Cortex Cerebellar Nuclei

124 ± 7 114 ± 7 198 ± 7 99 ± 3 141 ± 5 133 ± 4 69 ± 3 66 ± 2 106 ± 4

79 ± 5 75 ± 4 131 ± 8 59 ± 7 104 ± 7 81 ± 4 46 ± 3 44 ± 2 75 ± 4

−36 −34 −34 −40 −26 −39 −33 −33 −29

White Matter 42 ± 2 35 ± 5 35 ± 5 38 ± 2

30 ± 2 30 ± 2 29 ± 2 29 ± 2

−29 −14 −17 −24

Visual Cortex Auditory Cortex Parietal Cortex Sensory-motor Cortex

Corpus Callosum Genu of Corpus Callosum Internal Capsule Cerebellar White Matter

Note: The effects of anesthesia were statistically signiﬁcant in every structure (p < 0.05). From Sokoloff et al. (1977).

16

Brain Energetics and Neuronal Activity Superior colliculus 160 Stratum griseum superficiale

Local cerebral glucose utilization (µ mol/100 g/min)

140 120 100 80

Stratum lemnisci

60

Stratum opticum

40 Albino Pigmented

20 0.3 0

0

0.1

1.4 1

7

700

10

100

1000

70000 10000

Light intensity (lux) Lateral geniculate nucleus 160

Local cerebral glucose utilization (µ mol/100 g/min)

140

Dorsal nucleus

120 100 Ventral nucleus

80 60 40

Albino Pigmented

20 0.3 0

0

0.1

1.4 1

7

700

10 100 1000 Light intensity (lux)

70000 10000

Figure 2.2. Rates of glucose utilization as a function of intensity of retinal illumination with randomly timed light ﬂashes in various layers of superior colliculus and lateral geniculate nucleus of dark adapted albino and pigmented rats. From Miyaoka et al. (1979).

superior cervical ganglion (Yarowsky et al., 1983) and dorsal horn of the lumbar spinal cord, respectively (Figure 2.3) (Kadekaro et al., 1985). The glucose consumed per action potential can be determined from the slope of the straight line and equals 0.4 nmol/g of tissue per spike in the superior cervical ganglion and 0.6 nmol/g per spike in the dorsal horn of the lumbar spinal cord (Figure 2.3).

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States

17

Energy metabolism per spike Dorsal horn of lumbar spinal cord 1 0.8 Glucose utilization (µ mol/g/min)

0.6 0.4

Glucose consumption: 0.6 nmols/g per spike

0.2 0 0

5 10 Spike frequency (Hz)

15

Superior cervical ganglion 1 0.8 0.6 0.4

Glucose consumption: 0.4 nmols/g per spike

0.2 0 0

5 10 Spike frequency (Hz)

15

Figure 2.3. Relationship between frequency of action potentials and glucose utilization in dorsal horn of lumbar spinal cord and in superior cervical ganglion during electrical stimulation of sciatic nerve or cervical sympathetic trunk at different frequencies, respectively. Derived from data of Kadekaro et al. (1985) (top) and Yarowsky et al. (1983) (bottom).

2.3. BIOCHEMICAL MECHANISMS OF FUNCTIONAL ACTIVATION OF ENERGY METABOLISM AT THE TISSUE LEVEL Muscles move or support masses against gravitational forces; the heart pumps blood against a pressure head; and kidneys transport water and solutes against concentration and osmotic gradients. These functions involve clearly deﬁnable physical work that requires expenditure of energy. It was less obvious what energy-requiring work nervous tissues do when functionally activated. Inasmuch as CMRglc increases more or less linearly with spike frequency (Figure 2.3), it was reasonable to presume that the increased energy metabolism was somehow related to the generation, propagation, and conduction of action potentials. Action potentials result from the opening of ion channels that lead to increased Na+ inﬂux and K+ efﬂux in neurons; they do not themselves directly consume energy but draw upon the potential energy stored in the membrane potentials. As spike frequencies increase, there are greater ion displacements and larger rises in intracellular Na+ ([Na+ ]i ) and extracellular K+ ([K+ ]o ) concentrations and, therefore, the need for more electrogenic Na+ , K+ -ATPase activity to restore the ionic gradients across the membranes to their resting levels. This enzyme catalyzes the coupled transport of Na+ out of and K+ into the cells and consumes one molecule of ATP for every three Na+ ion exported and two K+ ions imported. Increased ATPase activity lowers intracellular ATP and raises ADP, AMP, phosphate acceptor, and inorganic phosphate concentrations, intracellular changes that stimulate glycolysis and electron transport and, therefore, energy metabolism. Many of the conditions of functional activation in which the [14 C]DG method revealed local increases in CMRglc have, in fact, been shown to be associated with elevated [K+ ]o in the activated tissues, and increasing. [K+ ]o in vivo by applying K+ directly to the cerebral cortex, a treatment known to depolarize cell membranes and to cause spreading cortical depression, which itself raises [K+ ]o even further, profoundly stimulates cerebral cortical glucose utilization (Figure 2.4) (Shinohara et al., 1979).

18

Brain Energetics and Neuronal Activity (A)

(B)

Frontal cortex Control

Sensory-motor cortex

During spreading depression

Parietal cortex

During recovery from spreading depression

Figure 2.4. [14 C]Deoxyglucose autoradiographs showing changes in local CMRglc during K+ -induced spreading cortical depression and during recovery in the rat; greater optical densities indicate higher CMRglc . In all cases the experimental side on the left was treated with topically applied KCl, and the control side on the right was similarly treated with equivalent concentrations of NaCl. (A) Autoradiographs of brain sections at various levels of cerebral cortex from conscious rat in which spreading cortical depression was produced and sustained on left side by 5 M KCl applied to surface of intact dura over parietal cortex every 15–20 min; right side treated comparably with NaCl. (B) Autoradiographs of brain sections at level of parietal cortex from three rats under barbiturate anesthesia. Top section is from normal, anesthetized rat; middle section is from similarly anesthetized rat in which 80 mM KCl in artiﬁcial cerebrospinal ﬂuid was repeatedly applied directly to the surface of the left parietal cortex; bottom section is from similarly anesthetized rat studied immediately after return of cortical DC potential to normal after a single wave of spreading depression induced by a single application of 80 mM KCl to the parieto-occipital cortex of the left side. From Shinohara et al. (1979).

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States

19

Table 2.3. Inﬂuence of sodium pump activity and neurosecretion on [14 C]deoxyglucose uptake in posterior pituitary in vitroa Condition

[14 C]Deoxyglucose uptake (Cpm/100 µg protein/15 min)

A. Dependence on activation of sodium pump Controls (4) + Electrical stimulation at 10 Hz (4) + Electrical stimulation at 10 Hz + 10 µM ouabain (4) B. Dependence on activation of sodium pump Controls (14) +60 µM veratridine (14) +6 µM tetrodotoxin (9) +60 µM veratridine + 6 µM tetrodotoxin (8) +10 µM ouabain (4) +60 µM veratridine + 10 µM ouabain (4) C. Independence from activation of secretion (Ca2+ -free medium) Controls (in Ca2+ -free medium) (6) +60 µM veratridine (in Ca2+ -free medium) (6)

Electrical stimulation 988 ± 19 1272 ± 57b 1018 ± 51c Opening Na+ channels 1381 ± 50 1891 ± 85b 1209 ± 84c 1551 ± 72c 1318 ± 57c 1218 ± 120c 1142 ± 38 1681 ± 78b

The values represent means ± SEM of results obtained in number of experiments indicated in parentheses. Indicates statistically signiﬁcant difference from controls (p < 0.001). c Indicates no statistical signiﬁcant difference from controls. From Mata et al. (1980). a b

The possibility that activation of Na+ , K+ -ATPase activity by membrane-depolarizing stimulation mediates the coupling of energy metabolism to functional activity was examined directly in studies in which the rat neurohypophysis was electrically stimulated in vitro under conditions known to stimulate its secretion of vasopressin (Mata et al., 1980). Such electrical stimulation raised [14 C]DG phosphorylation, indicating increased glucose utilization, and this stimulation was completely blocked by addition of ouabain, a speciﬁc inhibitor of Na+ , K+ -ATPase (Table 2.3). Addition of veratridine, an agent that depolarizes cell membranes by opening voltage-dependent Na+ channels and allows Na+ entry into the cells, also markedly stimulated [14 C]DG phosphorylation, and this stimulation too was eliminated by ouabain, as well as by tetrodotoxin, a known blocker of voltage-dependent Na+ channels (Table 2.3). The stimulation of [14 C]DG phosphorylation was not related to increased vasopressin secretion; the neurohypophysis in vitro cannot be stimulated to secrete hormone in Ca++ -free medium, but even in such a medium veratridine stimulated [14 C]DG phosphorylation (Table 2.3). It appears then that the extra energy metabolism associated with electrical and functional activation in neural tissue is not used directly in the generation and propagation of action potentials. The energy is used rather to restore ionic gradients and resting membrane potentials that are partially degraded by the action potentials and also to some extent for other processes associated with or secondary to action potentials, such as neurotransmitter release, reuptake, and metabolism, exocytosis, endocytosis, etc.

2.4. LOCALIZATION OF FUNCTIONALLY ACTIVATED ENERGY METABOLISM AT THE TISSUE LEVEL Traditional electrophysiology focuses largely on electrical recordings from neurons, and it was, therefore, originally assumed that the increases in local CMRglc associated with functional activation observed with the [14 C]DG method were primarily in perikarya. Careful comparisons between the autoradiographs produced by the [14 C]DG method and the stained brain sections from which they were prepared did not, however,

20

Brain Energetics and Neuronal Activity Molecular layer of hippocampus

Figure 2.5. [14 C]Deoxyglucose autoradiograph of normal, conscious rat brain. Greater optical density reﬂects greater CMRglc . Note high rate in molecular layer of hippocampus.

support this assumption. In the autoradiographs higher rates of glucose utilization are reﬂected in increasing darkness in the ﬁlms (Figure 2.1). It was noted in the autoradiographs that the most metabolically active region in the hippocampus, which was at ﬁrst thought to represent the cell-rich pyramidal cell layer, was actually the molecular layer, a region predominantly rich in synapses and poor in cell bodies (Figure 2.5). In the striate cortex of the normal monkey CMRglc is highest in a sub-layer of Layer IV that is not particularly rich in cellular elements; it is the layer where axonal terminals of the afferent geniculocalcarine pathway synapse with dendrites of neurons situated in other laminae of the visual cortex (Figure 2.6A, C) (Kennedy et al., 1976). It is in this neuropil-rich layer that CMRglc is most reduced when visual input is interrupted (Figure 2.6B). The hypothalamo-neurohypophysial pathway originates in cell bodies in the supraoptic and paraventricular nuclei in the hypothalamus and terminates in the neurohypophysis, 40 % of which consists of axonal terminals of the afferent tract (Nordmann, 1977). Osmotic stimulation by salt-loading is known to activate this pathway to stimulate vasopressin release from the neurohypophysis. Salt-loading was found also to stimulate local CMRglc markedly in the neurohypophysis but to have no apparent effect on CMRglc in the supraoptic and paraventricular nuclei (Figure 2.7) (Schwartz et al., 1979). In contrast, hypotension produced by α-adrenergic blockade with phenoxybenzamine or hemorrhage was found to stimulate local CMRglc markedly in both these nuclei (Figure 2.7). The difference is that osmotic stimulation acts directly on the cell bodies to activate the hypothalamo-neurohypophysial pathway whereas hypotension exerts its effects on the cell bodies in these nuclei indirectly via afferent inputs from the nucleus tractus solitarius in the brain stem that are part of the neural pathways of the baroreceptor reﬂexes. Results such as these suggested that it is mainly the energy metabolism in regions rich in neuropil and synapses and not cell bodies that is linked to functional activity. To compare directly the effects of functional activation on local CMRglc in perikarya and nerve terminals simultaneously in the same pathway, Kadekaro et al. (1985) stimulated the sciatic nerve at different frequencies and examined the effects on CMRglc in both the dorsal root ganglia and the dorsal horn of corresponding segments of the lumbar spinal cord. The advantage of this system is that the body of the dorsal root ganglion contains perikarya devoid of neural processes, thus allowing examination of cell bodies free of nerve terminals simultaneously with the nerve terminals of the same pathway in the dorsal horn of the spinal cord. The results conﬁrmed that it is CMRglc in the region of the nerve terminals and not in the cell bodies that is linked to the functional activity. Glucose utilization increased linearly with the

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States

21

(A)

(B)

(C)

Figure 2.6. [14 C]Deoxyglucose autoradiographs demonstrating effects of bilateral and monocular visual occlusion on local CMRglc in striate cortex of conscious monkey. Greater optical density indicates greater CMRglc . (A) Striate cortex from animal with both eyes open. Note heterogeneity in laminae; the darkest lamina corresponds to Layer IV. (B) Striate cortex from monkey with both eyes patched. Note general reduction in density and almost complete disappearance of laminar heterogeneity, especially in Layer IV. (C) Striate cortex from animal with only right eye patched. Left half of autoradiograph corresponds to left hemisphere contralateral to occluded eye. Note alternating dark and light columns traversing the full thickness of the striate cortex which represent the ocular dominance columns. The dark bands represent the columns for the open eye; the light bands represent the columns for the patched eye and demonstrate the reduced glucose utilization resulting from reduced visual input. The arrows point to regions of bilateral asymmetry; these are the loci of representation of the blind spots of the visual ﬁelds. From Kennedy et al. (1976).

increasing frequency of stimulation of the sciatic nerve in the dorsal horn of the lumbar spinal cord, but it did not budge from its baseline levels in the cell bodies of the same pathway in the dorsal root ganglia (Figure 2.8). The lack of a metabolic response to electrical stimulation in the perikarya in the dorsal root ganglion may be surprising, but there is evidence that the soma membrane of neurons is not very excitable and not very productive of action potentials because of a relative paucity of voltage-gated Na+ channels. Smith (1983b), using patch clamp electrodes, obtained evidence that soma and dendrites of spinal cord neurons and soma of dorsal root ganglion cells cultured in vitro do not generate action potentials. Freygang

22

Brain Energetics and Neuronal Activity (A)

PP

AP

PVN SON (B)

(C)

(D)

Figure 2.7. Effects of activation of hypothalamo-neurohypophysial pathway by salt-loading or hypotension on local cerebral glucose utilization in the conscious rat. (A) Histological sections of brain stained with cresyl violet (Nissl) and pituitary stained with toluidine blue demonstrating positions of supraoptic nucleus (SON), paraventricular nucleus (PVN), posterior pituitary (PP), and anterior pituitary (AP). (B) [14 C]Deoxyglucose autoradiographs of brain and pituitary from normal control rat drinking only water. (C) [14 C]Deoxyglucose autoradiographs from rat given 2 % (w/v) NaCl in drinking water for 5 days. Note selective marked increase in density in posterior hypophysis, indicating increased glucose utilization. (D) [14 C]Deoxyglucose autoradiographs from rat made hypotensive by administration of 20 mg/kg of phenoxybenzamine 45–60 min prior to administration of the [14 C]deoxyglucose. Note selective increases in labeling of supraoptic and paraventricular nuclei and posterior pituitary. From Schwartz et al. (1979).

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States

23

100

Glucose utilization (µ mol/100 g/min)

Spinal cord

(4)

80

(4) 60 (4) Means ± SEM (n) = Number of animals

40 (12)

Dorsal root ganglion 20

0 0

5 10 Frequency of stimulation (Hz)

15

Figure 2.8. Frequency-dependent effects of electrical stimulation of sciatic nerve on local CMRglc in dorsal root ganglion and dorsal horn of lumbar spinal cord. Error bars represent SEM. From Kadekaro et al. (1985).

(1958) and Freygang and Frank (1959) had previously concluded from analyses of extracellular potentials recorded from single spinal motor neurons and single neurons in the lateral geniculate nucleus that the soma-dendritic membrane can be driven synaptically to produce post-synaptic potentials but not propagating action potentials. Conduction through the cell body is mainly by electrotonic conduction rather than by action potentials. If action potentials do indeed mediate the coupling of energy metabolism to functional activity but are absent in the perikarya, then increased glucose metabolism due to functional activation is not to be expected in the perikarya. Some energy metabolism does, of course, proceed in cell bodies even at rest, but it is probably used more for vegetative, biosynthetic, and transport processes needed to maintain cellular structural and functional integrity rather than for processes directly related to functional activity. Some energy-consuming processes within the cell bodies that are related to intracellular signal transduction must also be activated by functional activation, but apparently their requirements are too small to be detected in the overall energy consumption of the cell. The ﬁnding that rates of energy metabolism are linked to spike frequency in the terminal zones of the afferent pathways in the neuropil and not in cell bodies in the pathway should help to resolve questions about metabolic responses associated with functional excitation and inhibition. Glucose utilization has sometimes been observed to increase in structures in which electrophysiological evidence indicated inhibition of neuronal activity, thus raising questions whether active inhibition required energy just like excitation. It appears, however, that it is the spike activity in the afferent nerve terminals that correlates with the energy consumption, and this activity is the same whether the terminals are releasing excitatory or inhibitory neurotransmitters. The energy metabolism of the post-synaptic cell bodies, whether activated or inhibited, is not signiﬁcantly altered, and to determine which has occurred, it is necessary to look downstream at the next synapses in the projection zones of those neurons. Glucose utilization is depressed in the projection zones of inhibited neurons and increased in the projection zones of excited neurons.

24

Brain Energetics and Neuronal Activity

2.5. CELLULAR LOCALIZATION OF FUNCTIONAL ACTIVATION OF ENERGY METABOLISM The neuropil, in which functionally activated energy metabolism is localized, contains not only neuronal elements, e.g., axonal terminals, dendritic processes, and synapses, but astrocytic processes as well. Because stimulation of local CMRglc is linked to Na+ , K+ -ATPase activity and the frequency of action potentials which are associated with increased Na+ inﬂux and K+ efﬂux, some of the increased energy metabolism must be used to restore the ionic gradients in the neuronal elements where the action potentials are produced. Astrocytic processes envelop the synapses and could also be involved in the metabolic response. Astrocytes are believed to regulate [K+ ]o either by passive diffusion (Orkand et al., 1966; Medzihradsky et al., 1971) and/or active transport (Henn et al., 1972) following increases in [K+ ]o resulting from neuronal excitation (Hertz, 1977; Erecinska and Silver, 1994), and studies with tissue slices (Yarowsky et al., 1986; Badar-Goffer et al., 1992) or cultured cells (Cummins et al., 1979a; Cummins et al., 1979b; Brookes and Yarowsky, 1985; Hertz and Peng, 1992; Peng et al., 1994) have indicated that energy is consumed in the process. The spatial resolution of the autoradiographic [14 C]DG method is limited to 100–200 µm (Smith, 1983a) which is inadequate to identify the speciﬁc cellular or subcellular elements in neuropil that contribute to the increases in CMRglc during functional activation.

2.5.1. Effects of Increased Extracellular K+ and Intracellular Na+ Concentrations To approach this question, studies have been carried out in vitro with cells in culture. Changes in the extracellular medium expected to result from increased spike activity in vivo were simulated in vitro, and their effects on glucose metabolism of cultured neurons and astroglia examined. Spike activity in axonal terminals leads to increased extracellular K+ and intracellular Na+ concentrations and release of neurotransmitters. Therefore, the effects of elevated [K+ ]o , [Na+ ]i , and the extracellular concentration of glutamate, the most prevalent excitatory neurotransmitter in brain, on [14 C]DG phosphorylation to [14 C]DG6-phosphate, a measure of glucose utilization, were examined in cultured neurons and astroglia (Takahashi et al., 1995). Increasing [K+ ]o in the range of 5.4–56 mM stimulated [14 C]DG phosphorylation in both neuronal and mixed neuronal-astroglial cultures (Figure 2.9) (Takahashi et al., 1995), and these increases were prevented by ouabain, implicating Na+ , K+ -ATPase in the mechanism of the effect. Raising [K+ ]o in the same range had no such effect in the cultured astroglia. In fact, the highest levels of [K+ ]o examined tended to inhibit [14 C]DG phosphorylation in astroglia (Figure 2.9). On the other hand, promoting Na+ entry into the cells and increasing [Na+ ]i either by opening membrane voltage-gated Na+ channels with veratridine or by adding the Na+ ionophore monensin markedly stimulated [14 C]DG phosphorylation in astroglia, but only if Na+ were present in the extracellular medium (Figure 2.10) (Brookes and Yarowsky, 1985; Takahashi et al., 1995). Tetrodotoxin, which blocks voltage-dependent Na+ channels, did not alter baseline rates of [14 C]DG phosphorylation in astroglia, and, as expected, prevented the stimulation by veratridine and had no effect on the stimulation by monensin (Figure 2.10). Inhibiting Na+ , K+ -ATPase activity with ouabain lowered the basal rate of [14 C]DG phosphorylation, completely blocked the stimulation by veratridine, and partially suppressed the stimulation by monensin (Figure 2.10). The absence of a tetrodotoxin effect on the monensin stimulation can be explained by the fact that monensin-induced Na+ entry is not through voltage-dependent Na+ channels and is, therefore, insensitive to blockade by tetrodotoxin. The less than complete blockade of the monensin stimulation by ouabain can also be explained by the fact that monensin is a Na+ /H+ exchanger that exchanges extracellular Na+ for intracellular H+ . It, therefore, raises Na+ and lowers H+ concentrations in the cells, both ionic changes that can enhance glucose metabolism, the

[14C]Deoxyglucose-6-phosphate (DPM/µg protein)

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States 140 120 100 80 60 40 20 0

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180 160 140 120 100 80 60 40 20 0

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30 min ** **

15 min ** ** 10 min *

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56

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30 min ** 15 min ** **

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28

56

30 min ** 15 min * **

10 min

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28

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28

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Extracellular potassium ion concentration (mM)

Figure 2.9. Effects of [K+ ]o on rates of [14 C]DG phosphorylation in cultured neurons, mixed neuronal-astroglia cultures, and astroglia cultures. Values are means ± SEM obtained from quadruplicate wells. Numbers above bars indicate duration of incubation. ∗ p < 0.05, ∗∗ p < 0.01 compared to the 5.4 mM [K+ ]o (Dunnett’s test for multiple comparisons). Representative of at least three such experiments for each condition. From Takahashi et al. (1995).

former by ouabain-sensitive stimulation of Na+ , K+ -ATPase activity, and the latter by ouabain-insensitive stimulation of phosphofructokinase activity due to increased intracellular pH.

2.5.2. Effects of Glutamate on Glucose Utilization in Astroglia Glutamate, the most prevalent excitatory neurotransmitter in the brain, is toxic to neurons, and must, therefore, be rapidly cleared from the extracellular space when it is released at glutamatergic synapses. Astrocytes, which have processes that envelop the synapses, carry out this function. They take up the glutamate from the extracellular space and convert it to glutamine, both steps that involve the consumption of energy. Addition of glutamate to the medium stimulates glucose utilization by astroglial cultures (Pellerin and Magistretti, 1994; Takahashi et al., 1995). This stimulation is Na+ dependent, ouabain sensitive, unaffected by tetrodotoxin, and insensitive to NMDA and non-NMDA glutamate receptor antagonists (Figure 2.11). DL-Threo-β-hydroxyaspartate, which competes with glutamate for uptake by

26

Brain Energetics and Neuronal Activity

[14C]Deoxyglucose-6-phosphate (DPM/µg protein)

Veratridine 120

No Veratridine

+ 20% **

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60

−5% n.s

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+Ouabain (1 mM)

+TTX (10 µM)

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Monensin

+ 189% ***

+ 171% ***

200 +124% ***

150 100

−26% **

50 0

Control

+Ouabain (1 mM)

+TTX (10 µM)

Na+free

Figure 2.10. Effects of veratridine (upper) and monensin (lower) on [14 C]DG phosphorylation in astroglia (21-day old culture, no dbcAMP) and the effects of ouabain, tetrodotoxin (TTX), and Na+ -free medium on the veratridine and monensin effects. Values are means ± SEM obtained from measurements of quadruplicate wells in three experiments. Numbers above bars indicate percent difference from each control. Representative of in three different astroglial preparations. ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not statistically signiﬁcant from each control (grouped t test). From Takahashi et al. (1995).

the Na+ /glutamate co-transport system (Flott and Seifert, 1991), itself stimulates glucose utilization, and glutamate no longer stimulates in its presence. The hydroxyaspartate stimulation is also blocked by ouabain, absent in Na+ -free medium, and unaffected by tetrodotoxin and inhibitors of NMDA or non-NMDA receptors. These results show that the glutamate stimulation of glucose utilization by astroglia does not involve NMDA and non-NMDA glutamate receptors or voltage-dependent Na+ channels in the astroglia. It is due instead to the activation of Na+ , K+ -ATPase activity by the increase in [Na+ ]i that results from the coupled uptake of Na+ with glutamate into the astroglia. This uptake is mediated by a Na+ /glutamate co-transporter that co-transports 2–3 Na+ ions with each glutamate molecule. In addition, much of the glutamate taken up by the astroglia is converted by glutamine synthetase to glutamine, a reaction consuming one ATP molecule for each molecule of glutamine formed. Inasmuch as it takes one molecule of ATP to pump out

Energy Metabolism in Neural Tissues in vivo at Rest and in Functionally Altered States 120

No Glutamate + 2% n.s

500 µM Glutamate [14C]Deoxyglucose-6-phosphate (DPM/µg protein)

27

100 + 44% ** *

+ 38% ** *

80

+ 50% ** *

+ 28% ** *

60

40 + 9% n.s

−19% ** * 20

0

Control + Ouabain +TTX (1 mM) (10 µM)

+APV (1 mM)

+CNQX (100 µM)

+THA (1 mM)

Na+free

Figure 2.11. Effects of glutamate on [14 C]DG phosphorylation in astroglia and the effects of DL-2-amino-5-phosphonovaleric acid (APV), CNQX, ouabain, tetrodotoxin (TTX), DL-threo-β-hydroxyaspartic acid (THA), and Na+ -free medium on the glutamate effects. Values are means ± SEM obtained from quadruplicate wells, in three experiments, each with a different astroglial preparation. Numbers above bars indicate percent differences from each control. ∗∗∗ p < 0.001; n.s., not statistically signiﬁcant, from each control group (grouped t test). From Takahashi et al. (1995). Similar results were reported by Pellerin and Magistretti (1994).

the 2–3 Na+ ions co-transported inward with each molecule of glutamate taken up, and one molecule of ATP is consumed in converting the molecule to glutamine, it appears that the net gain of two molecules of ATP from the glycolysis of one molecule of glucose is consumed in the processing of one molecule of glutamate by astroglia.

2.6. SUMMARY The main energy-consuming work of functionally activated neural tissues is related to the generation and propagation of action potentials and the disposition of the neurotransmitter molecules that are released at the synapses. Normally, the energy required by neural tissues is almost entirely derived from glucose metabolism which increases almost linearly with spike frequency when tissue is functionally activated. Spikes result from Na+ inﬂux and K+ efﬂux in the neuronal elements where they are generated, and the greater the spike frequency the greater the ionic displacements. Increased extracellular K+ and intracellular Na+ concentrations stimulate Na+ , K+ -ATPase activity to restore the ionic gradients to their normal resting levels, and energy metabolism is then increased to resynthesize the ATP consumed in that process. Functional activation of glucose utilization is essentially conﬁned to neuropil which contains axonal, dendritic, and astrocytic processes. Obviously, some of the increased energy metabolism must occur in the

28

Brain Energetics and Neuronal Activity

axonal terminals and dendrites where the spikes are generated. Functional activation of energy metabolism is not seen in perikarya, presumably because they have few voltage-dependent Na+ channels and, therefore, generate few if any action potentials. Astrocytes contribute to the increased energy metabolism in neuropil during functional activation, but by other mechanisms. Astrocytic membranes can be depolarized by increased [K+ ]i , but they do not produce action potentials that would allow the Na+ inﬂux into the cells needed to stimulate Na+ , K+ -ATPase activity. Action potentials in axonal terminals, however, are associated with release of neurotransmitters. Glutamate is the most prevalent excitatory neurotransmitter in brain, but it is also a neurotoxin, and its extracellular concentration must be kept low by astrocytic reuptake via Na+ -dependent glutamate transporters that transport 2–3 Na+ ions with each glutamate molecule into the cell. The increased [Na+ ]i then results in stimulation of Na+ , K+ -ATPase activity and energy metabolism. In addition, glutamate taken up by astroglia is converted there to glutamine, an energy-requiring reaction that also consumes ATP. Astrocytic processes surround the capillaries that bring glucose to the brain, and some if not all the glucose must traverse them before reaching the neurons. Evidence has been accumulating that astrocytes ﬁrst metabolize glucose to lactate which they then export to the neurons for oxidation to CO2 and H2 O (Magistretti and Pellerin, 1996; Tsacopoulos and Magistretti, 1996). This would mean that glucose metabolism is compartmentalized between astroglia and neuronal elements, glycolysis in the astroglia and oxidation of the lactate/pyruvate products by the neurons. Glycolysis yields two molecules of ATP and two molecules of pyruvate/lactate per molecule of glucose glycolyzed. The two molecules of ATP produced in the astroglia are then consumed in the processing of each molecule of glutamate, suggesting that there may be a close quantitative relationship between glucose consumption and glutamate processing in the astroglia (Sibson et al., 1998). The oxidation of the two molecules of pyruvate/lactate produced from glycolysis of one molecule of glucose leads to the generation of 36 additional molecules of ATP which is consumed mainly for the restoration of the ionic gradients and the membrane potentials degraded by the action potentials.

REFERENCES Badar-Goffer, R. S., Ben-Yoseph, O., Bachelard, H. S., and Morris, P. G. 1992. Neuronal–glial metabolism under depolarizing conditions. A 13 C-n.m.r. study. Biochem. J. 282: 225–230. Brookes, N., and Yarowsky, P. J. 1985. Determinants of deoxyglucose uptake in cultured astrocytes: the role of the sodium pump. J. Neurochem. 44: 473–479. Clarke, D. D., and Sokoloff, L. 1999. Circulation and energy metabolism of the brain. In: Siegel, G., Agranoff, B., Albers, R. W., and Fisher, S. (Eds): Basic Neurochemistry: Molecular, Cellular, and Medical Aspects, 6th edn, Chapter 31. Lippincott-Raven, Philadelphia, PA, pp. 637–669. Cummins, C. J., Glover, R. A., and Sellinger, O. Z. 1979a. Neuronal cues regulate uptake in cultured astrocytes. Brain Res. 170: 190–193. Cummins, C. J., Glover, R. A., and Sellinger, O. Z. 1979b. Astroglial uptake is modulated by extracellular K+ . J. Neurochem. 33: 779–785. Erecinska, M., and Silver, I. A. 1994. Metabolism and role of glutamate in mammalian brain. Prog. Neurobiol. 43: 37–71. Flott, B., and Seifert, W. 1991. Characterization of glutamate uptake in astrocyte primary cultures from rat brain. Glia 4: 293–304. Freygang, W. H., Jr. 1958. An analysis of extracellular potentials from single neurons in the lateral geniculate nucleus of the cat. J. Gen. Physiol. 41: 543–564. Freygang, W. H., Jr. and Frank, K. 1959. Extracellular potentials from single spinal motoneurones. J. Gen. Physiol. 42: 749–760. Freygang, W. H., Jr. and Sokoloff, L. 1958. Quantitative measurements of regional circulation in the central nervous system by the use of a radioactive inert gas. Adv. Biol. Med. Phys. 6: 263–279. Henn, F. A., Haljam¨ae, H., and Hamberger, A. 1972. Glial cell function: active control of extracellular K+ concentration. Brain Res. 43: 437–443.

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Hertz, L. 1977. Drug-induced alterations of ion distribution at the cellular level of the central nervous system. Pharmacol. Rev. 29: 35–65. Hertz, L., and Peng, L. 1992. Energy metabolism at the cellular level of the CNS. Can. J. Physiol. Pharmacol. 70 (Suppl.): S145–S157. Kadekaro, M., Crane, A. M., and Sokoloff, L. 1985. Differential effects of electrical stimulation of sciatic nerve on metabolic activity in spinal cord and dorsal root ganglion in the rat. Proc. Natl. Acad. Sci., USA 82: 6010–6013. Kennedy, C., Des Rosiers, M. H., Sakurada, O., Shinohara, M., Reivich, M., Jehle, J. W., and Sokoloff, L. 1976. Metabolic mapping of the primary visual system of the monkey by means of the autoradiographic [14 C]deoxyglucose technique. Proc. Natl Acad. Sci. USA 73: 4230–4234. Kety, S. S. 1950. Circulation and metabolism of the human brain in health and disease. Am. J. Med. 8: 205–217. Kety, S. S., and Schmidt, C. F. 1948. The nitrous oxide method for the quantitative determination of cerebral blood ﬂow in man: Theory, procedure, and normal values. J. Clin. Invest. 27: 476–483. Krebs, H. A., Williamson, D. H., Bates, M. W., and Hawkins, R. A. 1971. The role of ketone bodies in caloric homeostasis. Adv. Enzyme Regul. 9: 387–409. Landau, W. M., Freygang, W. H., Rowland, L. P., Sokoloff, L., and Kety, S. S. 1955. The local circulation of the living brain; values in the unanesthetized and anesthetized cat. Trans. Am. Neurol. Assoc. 80: 125–129. Magistretti, P. J., and Pellerin, L. 1996. Cellular bases of brain energy metabolism and their relevance to functional brain imaging: Evidence for a prominent role of astrocytes. Cerebral Cortex 6: 50–61. Mata, M., Fink, D. J., Gainer, H., Smith, C. B., Davidsen, L., Savaki, H., Schwartz, W. J., and Sokoloff, L. 1980. Activity-dependent energy metabolism in rat posterior pituitary primarily reﬂects sodium pump activity. J. Neurochem. 34: 213–215. Medzihradsky, F., Nandhasri, P. S., Idoyaga-Vargas, V., and Sellinger, O. Z. 1971. A comparison of ATPase activity of the glial cell fraction and the neuronal perikaryal fraction isolated in bulk from rat cerebral cortex. J. Neurochem. 18: 1599–1603. Miyaoka, M., Shinohara, M., Batipps, M., Pettigrew, K. D., Kennedy, C., and Sokoloff, L. 1979. The relationship between the intensity of the stimulus and the metabolic response in the visual system of the rat. In: Cerebral Blood Flow and Metabolism. Gotoh, F., Nagai, H., and Tazaki, Y. (Eds). Hidejimi Seihanjo, Tokyo (Also, Acta Neurol. Scand., Suppl. 72, 60: 16–17). Nordmann, J. J. 1977. Ultrastructural morphometry of the rat neurohypophysis. J. Anat. 123: 213–218. Orkand, R. K., Nicholls, J. G., and Kufﬂer, S. W. 1966. Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia. J. Neurophysiol. 29: 788–806. Owen, O. E., Moran, A. P., Kemp, H. G., Sullivan, J. M., Herrera, M. G., and Cahill, G. F. 1967. Brain metabolism during fasting. J. Clin. Invest. 46: 1589–1595. Pellerin, L., and Magistretti, P. J. 1994. Glutamate uptake into astrocytes stimulates aerobic glycolysis: A mechanism coupling neuronal activity to glucose utilization. Proc. Natl Acad. Sci. USA 91: 10625–10629. Peng, L., Zhang, X., and Hertz, L. 1994. High extracellular potassium concentrations stimulate oxidative metabolism in a glutamatergic neuronal culture and glycolysis in cultured astrocytes but have no stimulatory effect in a GABAergic neuronal culture. Brain Res. 663: 168–172. Phelps, M. E., Huang, S. C., Hoffman, E. J., Selin, C., Sokoloff, L., and Kuhl, D. E. 1979. Tomographic measurement of local cerebral glucose metabolic rate in humans with (F-18)2-ﬂuoro-2-deoxy-d-glucose: validation of method. Ann. Neurol. 6: 371–388. Reivich, M., Kuhl, D., Wolf, A., Greenberg, J., Phelps, M., Ido, T., Cassella, V., Fowler, J., Hoffman, E., Alavi, A., Som, P., and Sokoloff, L. 1979. The [18 F]ﬂuoro-deoxyglucose method for the measurement of local cerebral glucose utilization in man. Circulation Res. 44: 127–137. Schwartz, W. J., Smith, C. B., Davidsen, L., Savaki, H., Sokoloff, L., Mata, M., Fink, D. J., and Gainer, H. 1979. Metabolic mapping of functional activity in the hypothalamo. Science 205: 723–725. Shinohara, M., Dollinger, B., Brown, G., Rapoport, S., and Sokoloff, L. 1979. Cerebral glucose utilization: Local changes during and after recovery from spreading cortical depression. Science 203: 188–19. Sibson, N. R., Dhankhar, A., Mason, G. F., Rothman, D. L., Behar, K. L., and Shulman, R. G. 1998. Proc. Natl Acad. Sci. USA 95: 316–321.

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Smith, C. B. 1983a. Localization of activity-associated changes in metabolism of the central nervous system with the deoxyglucose method: Prospects for cellular resolution. In: Barker, J. L., and McKelvy, J. F. (Eds): Current Methods in Cellular Neurobiology, Vol. I, Anatomical Techniques. Wiley, New York, pp. 269–317. Smith, T. G, Jr. 1983b. Sites of action potential generation in cultured neurons. Brain Res. 288: 381–383. Sokoloff, L. 1981. Localization of functional activity in the central nervous system by measurement of glucose utilization with radioactive deoxyglucose. J. Cereb. Blood Flow Metab. 1: 7–36. Sokoloff. L., Reivich, M., Kennedy, C., Des Rosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O., and Shinohara, M. 1977. The [14 C]deoxyglucose method for the measurement of local cerebral glucose utilization: Theory, procedure, and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28: 897–916. Takahashi, S., Driscoll, B. F., Law, M. J., and Sokoloff, L. 1995. Role of sodium and potassium in regulation of glucose metabolism in cultured astroglia. Proc. Natl Acad. Sci. USA 92: 4616–4620. Tsacopoulos, M., and Magistretti, P. 1996. Metabolic coupling between glia and neurons. J. Neurosci. 16: 877–885. Yarowsky, P., Boyne, A. F., Wierwille, R., and Brookes, N. 1986. Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry. J. Neurosci. 6: 859–866. Yarowsky, P., Kadekaro, M., and Sokoloff, L. 1983. Frequency-dependent activation of glucose utilization in the superior cervical ganglion by electrical stimulation of cervical sympathetic trunk. Proc. Natl Acad. Sci. USA 80: 4179–4183.

3 Techniques–MRS, fMRI, 13C NMR, Indirect Detection of 13C Robin de Graaf Department of Diagnostic Radiology, Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

3.1 Introduction 3.2 In vivo NMR Spectroscopy 3.2.1 Proton NMR 3.2.2 Phosphorus NMR 3.2.3 Carbon-13 NMR 3.3 Elementary Experimental Techniques 3.3.1 Spatial Localization 3.3.2 Shimming 3.3.3 Water Suppression 3.3.4 Signal Quantiﬁcation 3.4 Advanced Experimental Techniques 3.4.1 Introduction to Spectral Editing 3.4.2 Homonuclear Spectral Editing 3.4.3 Heteronuclear Spectral Editing

31 33 33 34 37 39 39 40 42 43 45 45 46 49

3.1. INTRODUCTION Nuclear magnetic resonance (NMR) is based on the magnetic properties of nuclei. When placed in a (strong) external magnetic ﬁeld, the nuclei can be observed by the absorption and emission of electromagnetic radiation. Purcell, Torrey and Pound (1) at MIT, Cambridge and Bloch, Hansen and Packard (2) at Stanford Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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Brain Energetics and Neuronal Activity

simultaneously, but independently, discovered NMR in 1945. In 1952 Bloch and Purcell shared the Nobel prize for physics in recognition of their pioneering achievements (1–4). At this stage, NMR was purely an experiment for physicists to determine the nuclear magnetic moments of nuclei. NMR could only develop to one of the most versatile forms of spectroscopy after the discovery that nuclei within the same molecule absorb energy at different resonance frequencies. These so-called chemical shift effects, which are directly related to the chemical environment of the nuclei, were ﬁrst observed in 1949 by Proctor and Yu (5), and independently by Dickinson (6). During the ﬁrst two decades, NMR spectra were recorded in a continuous wave mode in which either the magnetic ﬁeld strength or the radio frequency was sweeped through the spectral area of interest, whilst keeping the other ﬁxed. In 1966, NMR was revolutionized by Ernst and Anderson (7) who introduced pulsed NMR in combination with Fourier transformation, leading to greatly enhanced sensitivity and increased versatility (e.g. multi-pulse experiments). Pulsed or Fourier transform NMR is at the heart of all modern NMR experiments. The induced energy level difference of nuclei in an external magnetic ﬁeld is very small when compared to the thermal energy, so that the energy levels are almost equally populated. As a result the absorption of photons (electromagnetic quanta) is very low, making NMR a very insensitive technique when compared to other forms of spectroscopy. However, as a consequence of the minimal perturbation of the spins by the external magnetic ﬁeld, NMR is also a noninvasive and nondestructive technique, ideally suited for in vivo measurements. In fact, by observing the water signal from his own ﬁnger, Bloch was the ﬁrst to use NMR on a living system. Soon after the discovery of NMR, others showed the possibility of NMR to study living objects. In 1950, Shaw and Elsken (8) used proton NMR to investigate the water content of vegetable material. Odebald and Lindstrom (9) obtained proton NMR signals from a number of mammalian preparations in 1955. Continued interest in deﬁning and explaining the properties of water in biological tissues led to the promising report of Damadian in 1971 (10) that NMR properties (relaxation times) of malignant tumorous tissues signiﬁcantly differ from normal tissue, suggesting that (proton) NMR may have diagnostic value. In the early 1970s, the ﬁrst high resolution NMR experiments on intact living systems were reported. Moon and Richards (11) used 31 P NMR on intact red blood cells and showed how the intracellular pH can be determined from chemical shift differences. In 1974, Hoult et al. (12) reported the ﬁrst study of 31 P NMR on intact, excised rat hindleg skeletal muscle. Four years later, the ﬁrst 31 P NMR studies on excised and in vivo mammalian brain were performed by Chance et al. (13) and improved with surface coil detection by Ackerman et al. (14) in 1980. Technical difﬁculties, such as water suppression, delayed the application of 1 H MRS to the study of brain metabolism. The ﬁrst in vivo 1 H MRS spectrum from rat brain was obtained by Behar et al. (15) in 1983 at Yale University. Two years earlier the same group also performed the ﬁrst in vivo 13 C MRS study on rat brain (16). Around the same time that reports on in vivo NMR spectroscopy appeared, Lauterbur (17) and somewhat later Mansﬁeld (18) described the ﬁrst reports on a major constituent of modern NMR, namely in vivo NMR imaging or magnetic resonance imaging (MRI). By applying position dependent magnetic ﬁelds in addition to the static magnetic ﬁeld, they were able to reconstruct the spatial distribution of the spins in the form of an image. In vivo NMR spectroscopy or magnetic resonance spectroscopy (MRS) and MRI have evolved from relatively simple one- or two-RF pulse sequences to complex techniques involving spatial localization, water and lipid suppression and spectral editing for MRS and time-varying magnetic ﬁeld gradients, ultrafast and multi-parametric acquisition schemes for MRI. Apart from MRI and MRS, there are many other tools available to study the anatomy, dynamics and metabolism of intact living tissues. For example, X-ray and computed tomography (CT) can provide high-resolution structural images, whereas others, including positron emission tomography (PET) and single photon emission computed tomography (SPECT) give rise to relatively low-resolution functional

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images. MRI has been shown to provide both high-resolution, high-contrast morphological images and high-resolution functional data. Furthermore, NMR does not require the use of ionizing radiation, making MRI a completely noninvasive and nondestructive imaging modality. The major advantage of in vivo NMR spectroscopy over the other mentioned modalities is, besides the noninvasive character, the excellent chemical speciﬁcity as expressed in the chemical shift and J coupling constants. This allows the study of speciﬁc metabolites and metabolic pathways, thereby offering an unique tool to study in vivo metabolism. This chapter will give a brief introduction to in vivo NMR spectroscopy with an emphasis on spectral appearance and experimental techniques. It does not represent a complete review, for which the interested reader is referred to the literature (19). It is assumed that the reader is familiar with the basic concepts of (organic) NMR spectroscopy.

3.2. IN VIVO NMR SPECTROSCOPY 3.2.1. Proton NMR The proton nucleus is, besides the low abundance hydrogen isotope tritium, the most sensitive nucleus for NMR, both in terms of intrinsic NMR sensitivity (high gyromagnetic ratio) and high natural abundance (>99.9 %). Since nearly all metabolites contain protons, in vivo 1 H MRS is, in principle, a powerful technique to observe, identify and quantify a large number of biologically important metabolites in intact tissue. However, the application of 1 H MRS to intact tissues in vivo is challenging for a number of reasons. Firstly, the water resonance is several orders of magnitude larger than the low-concentration metabolites, making metabolite detection difﬁcult and ambiguous. Therefore, water suppression is a prerequisite for meaningful in vivo 1 H MRS studies. More details on water suppression are given in Section 3.3.2. Secondly, the chemical shift range of in vivo 1 H NMR spectra is narrow, spanning approximately 5 ppm (for nonexchangeable protons). This causes a large number of metabolite resonances to overlap, making their detection and quantiﬁcation difﬁcult. Thirdly, even though the proton nucleus is the most sensitive for NMR studies, (in vivo) NMR is in general a very insensitive technique, making the detection of low-concentration metabolites a compromise between time resolution and signal-to-noise ratio. 3.2.1.1. Identiﬁcation of resonances The sensitivity of in vivo proton NMR typically limits the detection to compounds with a concentration higher than ∼0.5 mM. Despite this inherent limitation, an in vivo 1 H NMR spectrum still holds many resonances from a wide range of different metabolites. Figure 3.1 shows a typical in vivo 1 H NMR spectrum obtained from rat brain at 7 T. The dominating resonances are from the N(CH3 )3 group of total choline (Cho, ∼3.22 ppm) which consists mainly of phosphoryl choline and glycerophosphoryl choline; the CH3 group of total creatine (tCr, 3.03 ppm), consisting of creatine and phosphocreatine; and the CH3 groups of the combined N -acetyl resonances, mainly consisting of N -acetyl asparate (NAA, 2.01 ppm) with a smaller contribution from N -acetylaspartyl glutamate (NAAG, 2.04 ppm). In many clinical examinations performed at longer echo times, only the resonances from Cho, tCr and NAA are observed. However, in short TE spectra (Figure 3.1) many additional NMR resonances can be observed (Table 3.1), in particular myoinositol (mI) and glutamate (Glu). Even though these metabolites are present at similar concentrations as NAA (Table 3.1), they are less frequently observed since they are split into many smaller resonances due to scalar coupling interactions (see Section 3.4.1). Besides the high concentration metabolites, a typical short TE spectrum from brain holds information on alanine (Ala), aspartate (Asp), γ -aminobutyric acid (GABA), glucose (Glc), glutamine (Gln), glycine (Gly), lactate (Lac), scyllo-inositol (sI) and taurine (Tau). Typically the metabolite resonances are superimposed on a intense baseline arising from macromolecules (20, 21).

34

Brain Energetics and Neuronal Activity PCr/Cr NAA

Cho

PCr/Cr

Glu Gln NAAG

MM GABA GSH

Glu Gln

Glu

mI Tau

Gln Lac mI

NAA

Water

GABA

Asp

MM

Glc

5.0

4.0

MM MM

3.0

2.0

1.0

0.0

PPM

Figure 3.1. Proton NMR spectrum from rat brain in vivo (100 µl, STEAM localization, TR = 4000 ms, TE = 8 ms, TM = 25 ms, NEX = 128). Abbreviations are given for aspartate (Asp), choline-containing compounds (Cho), creatine (Cr), γ -aminobutyric acid (GABA), glucose (Glc), glutamate (Glu), glutamine (Gln), macromolecules (MM), myo-inositol (mI), N-acetyl aspartate (NAA), N-acetylaspartyl glutamate (NAAG), lactate (Lac), phosphocreatine (PCr) and taurine (Tau).

A detailed discussion on the biological function and signiﬁcance of the detected metabolites will be given in the following chapters. Table 3.1 gives a short overview of the known functions of the detected metabolites. The interested reader can ﬁnd additional information in (19, 22).

3.2.2. Phosphorus NMR The success of in vivo proton NMR spectroscopy in routine (clinical) MR is only matched by phosphorus NMR. The relatively high sensitivity of phosphorus NMR (circa 7 % of protons), together with a 100 % natural abundance allows the acquisition of high-quality spectra within minutes. Furthermore, the chemical shift dispersion of the phosphates found in vivo is relatively large (∼30 ppm), resulting in excellent spectral resolution even at low (clinical) magnetic ﬁeld strengths. Phosphorus NMR is very useful because with simple NMR methods it is capable of detecting all metabolites that play key roles in tissue energy metabolism. Furthermore, biologically relevant parameters such as intracellular pH may be indirectly deduced. 3.2.2.1. Identiﬁcation of resonances A typical in vivo phosphorus NMR spectrum holds a limited number of resonances (Figure 3.2). The exact chemical shift position of almost all resonances is sensitive to physiological parameters such as intracellular pH and ionic (magnesium) strength. By convention, the phosphocreatine resonance is used as

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Table 3.1. Proton chemical shifts, concentration range and biological function or methodological signiﬁcance for low molecular weight brain metabolitesa,b Compound

1

Acetate N -Acetyl aspartate N -Acetylaspartyl glutamate Alanine γ -Aminobutyric acid Aspartate Choline (total) Creatine Glucose

Glutamate Glutamine Glutathioned

Glycerophosphoryl choline

Glycine myo-Inositol scyllo-Inositol Lactate Phosphocreatine Phosphoryl choline Phosphoryl ethanolamine Taurine Threonine Valine a

H shift (ppm)

Resonance

Concentration range (mmol/kgww )

1.90 2.01 4.38, 2.67, 2.48 2.04

2

CH3 CH3 2 CH,3 CH2 ,3 CH2 2 CH3

0.4–0.8 7.9–16.6

3.77, 3.01, 3.89, 3.19, 3.03, 5.22, 3.52, 3.40, 3.83, 3.74, 2.34, 3.76, 2.43, 3.77 4.56, 3.77, 2.51, 3.61,

1.47 1.89, 2.80, 4.05, 3.91 4.63 3.23, 3.39, 3.88, 2.04, 2.35 2.13, 2.45

2

0.2–1.4 1.3–1.9 1.0–1.4 0.9–2.5 5.1–10.6 1.0

3.90, 4.31, 3.21 3.55 3.52, 3.61, 3.34 4.10,

3.87, 3.95 3.66

2.28 2.65 3.50

3.70, 3.47 3.82, 3.45 3.75, 3.71 2.12 2.11

2.93, 2.97 2.16, 2.15 2.56 3.67

2

CH,3 CH3 CH2 ,3 CH2 ,4 CH2 2 CH,3 CH2 ,3 CH2 N(CH3 )3 ,1 CH2 ,2 CH2 N(CH3 ),2 CH2 1 CH(α, β) 2 CH(α, β),3 CH(α, β) 4 CH(α, β),5 CH(α, β) 6 CH(α, β),6 CH(α, β) 2 CH,3 CH2 ,3 CH2 4 CH2 ,4 CH2 2 CH,3 CH2 ,3 CH2 4 CH2 ,4 CH2 Glycine moiety Cysteine moiety Glutamate moiety 2

1

Substrate of glial metabolism Neuronal marker Osmolite Involved in excitatory neurotransmission Meningioma marker Inhibitory neurotransmitter Excitatory neurotransmitter Membrane constituent Part of energy metabolism Substrate of energy metabolism Synthetic precursor

6.0–12.5 3.0–5.8 2.0

Excitatory neurotransmitter Intermediary metabolite Ammonia detoxiﬁcation Intermediary metabolite Antioxidant Storage of cysteine

1.0

Membrane constituent

0.4–1.0 3.8–8.1

1.31

CH,3 CH2 (glycerol) CH2 ,8 CH2 (choline) N(CH3 )3 2 CH2 1 CH,2 CH,3 CH 4 CH,5 CH,6 CH 1−6 CH 2 CH,3 CH3

3.03, 3.93 3.21, 4.28, 3.64 3.98, 3.22

N(CH3 ),2 CH2 N(CH3 )3 ,1 CH2 ,2 CH2 1 CH2 ,2 CH2

3.2–5.5 0.6 1.1–1.5

Inhibitory neurotransmitter Osmolyte Glial marker Unknown End product of anaerobic glycolysis Part of energy metabolism Membrane constituent Membrane constituent

3.42, 3.58, 3.60, 1.03,

1

0.9–1.5 0.3 0.1

Osmoregulator, neuromodulator Unknown Protein synthesis

4.05, 3.52 3.27, 3.61

3.25 4.25, 1.32 2.26 0.98

CH2 (glycerol)

0.6–2.7

Function/ signiﬁcancec

2 7

CH2 ,2 CH2 CH,3 CH,4 CH3 2 CH,3 CH 4 CH3 ,4 CH3 2

0.3–0.6 0.3–0.8

Chemical shifts are reported relative to the singlet resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS, also known as TSP) at 0.00 ppm. Water-exchangeable amide protons are not included. b The absolute concentration ranges were adapted from Govindaraju et al. c The function of many metabolites is only partially known and is the subject of ongoing research. d In vivo glutathione is almost completely present in the reduced GSH form.

36

Brain Energetics and Neuronal Activity PCr

ATP

PDE PME

Pi

g

a NADH

20

10

0

–10

b

–20

–30

PPM

Figure 3.2. Phosphorus NMR spectrum from rat brain in vivo (300 µl, ISIS localization, TR = 6000 ms, NEX = 128). Abbreviations are given for adenosine triphosphate (ATP), inorganic phosphate (Pi ), nicotinamide adenine dinucleotide (NADH), phosphocreatine (PCr), phospho-diesters (PDE) and phospho-monoesters (PME). Table 3.2. Phosphorus chemical shifts for low molecular weight metabolitesa Compound Adenosine monophosphate (AMP) Adenosine diphosphate (ADP) α β Adenosine triphosphate (ATP) α β γ Dihydroxyacetone phosphate Fructose-6-phosphate Glucose-1-phosphate Glucose-6-phosphate Glycerol-1-phosphate Glycerol-3-phosphorylcholine Glycerol-3-phosphorylethanolamino Inorganic phosphate Nicotinamide adenine dinucleotide (NADH) Phosphocreatine Phosphoenolpyruvate Phosphorylcholine

31

P chemical shift (ppm) 6.33 −7.05 −3.09 −7.52 −16.26 −2.48 7.56 6.64 5.15 7.20 7.02 2.76 3.20 5.02 −8.30 0.00 2.06 5.88

a Chemical shifts are reported relative to phosphocreatine at 0.00 ppm at pH 7.2 (fully complexed with magnesium).

an internal chemical shift reference and has been assigned a chemical shift of 0.00 ppm. At a pH of 7.2, with full magnesium complexation, the resonance of adenosine triphosphate (ATP) appear at −7.52 ppm (α), −16.26 ppm (β) and −2.48 ppm (γ ). The resonance of inorganic phosphate appears at 5.02 ppm. Under favorable, high-sensitivity conditions phosphorus NMR spectra can also hold resonances from phosphomono-esters and di-esters. Table 3.2 summarizes the chemical shifts of the most commonly observed 31 P-containing metabolites.

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3.2.2.2. Intracellular pH The chemical shift of many phosphorus containing compounds is dependent on a number of physiological parameters, in particular intracellular pH and magnesium concentration. The cause of this phenomenon can be found in the fact that protonation (or complexation with magnesium) of a compound changes the chemical environment of nearby nuclei and hence changes the chemical shift of those nuclei. When the chemical exchange between the protonated and unprotonated forms is slow, the two forms will have two separate resonance frequencies with the resonance amplitudes indicating the relative amounts of the two forms. However, for most compounds observed with phosphorus NMR, the chemical exchange is fast relative to the NMR time scale and only a single, average resonance is observed. The resonance frequency is now indicative of the relative amounts of protonated and unprotonated form, and hence the pH, and can be described by a modiﬁed Henderson–Hasselbach relationship according to δ − δHA pH = pKA + log (3.1) δA − δ where δ is the observed chemical shift, δA and δHA the chemical shifts of the unprotonated and protonated forms of compound A and pKA the logarithm of the equilibrium constant for the acid–base equilibrium between HA and A. Even though almost all resonances in 31 P NMR spectra have a pH dependence, the resonance of inorganic phosphate is most commonly used for several reasons. Its pK is in the physiological range (pK = 6.77), it is readily observed in most tissues (with muscle being a possible exception) and it has a large dependence on pH. Following similar arguments as outlined for intracellular pH, the free magnesium concentration can be deduced from the chemical shifts of ATP. 3.2.2.3. Creatine kinase The application of magnetization transfer to phosphorus NMR has allowed the non-invasive measurement of the creatine kinase (CK) reaction − MgATP2− + Cr PCr2− + MgADP− + H+ ← −−− −− →

(3.2)

The most generally accepted function of the CK/PCr system is that of ‘temporal energy buffering’, i.e. during a high workload the levels of ATP remain constant through the conversion of PCr (which is present at relatively high concentrations) to ATP. Other, more controversial functions include that of an ‘energy transport system’ and preventing the inactivation of ATPases by maintaining low intracellular ADP levels. The technique of magnetization transfer allows the determination of the absolute ﬂux through the CK enzyme by following the fate of labeled (i.e. saturated or inverted) magnetization from either PCr or γ -ATP. The forward ﬂux, from PCr to ATP is a relatively straightforward measurement. However, the measurement of the reverse ﬂux is complicated by ATP hydrolysis reactions by enzymes other than CK. However, proper modiﬁcations of the magnetization transfer experiment (i.e. multiple saturations) do allow the correct measurement of the reverse ﬂux (23). It is generally assumed that in skeletal muscle, heart and brain the creatine kinase catalyzed reactions are near or at equilibrium. Under this assumption, the intracellular ADP concentration can be calculated from Equation (3.2) if the equilibrium constant, intracellular metabolite (ATP, PCr, Cr) concentrations and pH are known.

3.2.3. Carbon-13 NMR Phosphorus and proton NMR spectroscopy have been successfully employed in a range of (clinical) in vivo NMR studies. Nevertheless, both nuclei have inherent limitations. Phosphorus NMR spectrum are normally

38

Brain Energetics and Neuronal Activity Glu C4

* 13C - 13C isotopomers

Glu C3 Glu C2 Gln C2 *

NAA C2 Cr C4 GPC C5

GABA C2

GABA C4 NAA C3 Asp C3 Cr C2

Asp C2

60

*

50

40

*

Gln C4

Gln C3 NAA C6 * Lac C3 GABA C3

*

30

20

PPM

Figure 3.3. Carbon-13 NMR spectrum from rat brain in vivo (500 µl, ISIS localization with polarization transfer, TR = 4000 ms, NEX = 512), averaged between 60 and 90 min following the intravenous infusion of [1, 6-13 C2 ]-glucose. Abbreviations are deﬁned in the legend of Figure 3.1. GPC = glycerophosphoryl choline.

characterized by a limited number of metabolites, while proton NMR is technically more challenging (e.g. water suppression) and suffers from an inherently poor spectral resolution. Carbon-13 NMR can offer complementary information to that obtained with phosphorus and proton NMR. Since (almost) all biologically relevant metabolites contain carbon, carbon-13 NMR is in principle capable of detecting many metabolites. Furthermore, the chemical shift dispersion extends over 200 ppm. However, the common carbon-12 isotope is not NMR active. Carbon-13 does have a magnetic moment, but is only present at a 1.1 % natural abundance. This, in combination with the low gyromagnetic ratio, makes carbon-13 NMR an inherently insensitive technique. Furthermore, strong heteronuclear scalar coupling interactions complicate the spectra and further reduce the sensitivity, such that double-resonance techniques (and hence additional, nonstandard hardware) must be employed to remove the effects of heteronuclear scalar coupling. Nevertheless, natural abundance carbon-13 NMR has been used to detect a wide range of metabolites, with a special emphasis on glycogen at 100.5 ppm. The low natural abundance of carbon-13 can be transformed into an advantage in that 13 C-enriched precursors can be infused to study metabolic pathways with little background interference from endogenous metabolites. Figure 3.3 shows a carbon-13 NMR spectrum from rat brain, 60 to 90 min after intravenous infusion of [1, 6-13 C2 ]-glucose. The 13 C label has been transferred from [1, 6-13 C2 ]-glucose to [4-13 C]-glutamate (34.2 ppm) by the action of the glycolytic pathway and the TCA cycle. The excitatory neurotransmitter glutamate is released into the synaptic cleft by exocytosis, interacts with post-synaptic receptors after which it is taken up by nearby astrocytes. There the glutamate is converted to glutamine, leading to the formation of [4-13 C]-glutamine (31.7 ppm). Other metabolic pathways lead to the labeling of [2-13 C]-GABA (35.2 ppm) and a range of other metabolites (chemical shifts are reported in Table 3.3). Using a mathematical model of compartmentalized

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Carbon-13 chemical shifts for low molecular weight metabolitesa 13

Compound

Acetate Alanine Aspartate Citrate Creatine Ethanol γ -Aminobutyric acid (GABA) Glycerol β-Hydroxybutyrate α-D-Glucose β-D-Glucose Glutamate Glutamine Glutathione (GSH, reduced) Glycerophosphorylcholine Glycine Glycogen Lactate myo-Inositol N-Acetyl aspartate Phosphorylethanolamine Taurine

C1

C2

182.6 176.6 175.1 179.7 175.4 17.9 182.3 63.6 181.2 92.7 96.6 175.3 174.8 56.6 62.9 173.3 100.5 183.3 73.3 179.7 61.9 48.4

24.5 51.5 53.0 46.8 37.8 58.4 35.2 73.3 47.6 72.1 79.9 55.5 55.0 55.1 71.5 42.5 72.0 69.3 73.1 54.0 41.6 36.2

C chemical shift (ppm) C3

C4

17.1 37.4 76.0 158.0

178.4 82.8 54.7

24.6 63.6 66.8 73.5 76.5 27.8 27.1 44.3 66.8 74.0 21.0 73.3 40.3

C5

C6

22.9 70.4 70.4 34.2 31.7 32.2 60.3

72.1 76.5 182.0 178.4 27.1 54.8

61.4 61.4

78.1

72.1

61.4

71.9 179.7

75.1 174.3

71.9 22.8

40.2

26.6

a

Chemical shifts are reported relative to the singlet resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS, also known as TSP) at 0.00 ppm.

brain energy metabolism, the formation of [4-13 C]-glutamate, [2-13 C]-GABA and [4-13 C]-glutamine can provide quantitative ﬂux information about the glutamatergic and GABAergic neuronal TCA cycles and the glutamate/GABA–glutamine neurotransmitter cycles between neurons and astrocytes. Metabolic modeling will be discussed in detail in Chapter 4. Besides the TCA cycle and related anaeplerotic pathways, 13 C NMR studies have been reported for a wide variety of other metabolic pathways, including gluconeogenesis (24, 25) and the synthesis and degradation of glycogen (26).

3.3. ELEMENTARY EXPERIMENTAL TECHNIQUES 3.3.1. Spatial Localization One of the most striking differences between high-resolution liquid-state NMR and in vivo NMR is that biological samples can be highly inhomogeneous in terms of metabolic composition as well as (B0 and B1 ) magnetic ﬁeld strengths and relaxation and diffusion parameters. Figure 3.4(B) shows a 1 H NMR spectrum of rat brain obtained with surface coil localization. The surface coil, as placed on top of the rat head, provides an inherent degree of spatial localization, limiting the sensitive volume to large parts of the brain with smaller contributions from skin, skull and muscle (Figure 3.4(A)). The 1 H NMR spectrum is characterized by two main signals arising from water (at ∼4.7 ppm) and methyl and methylene protons of fat and lipids (at ∼1.5 ppm). The resonance lines are generally very broad due to the inhomogeneous magnetic ﬁeld over the sensitive volume of the surface coil.

40

Brain Energetics and Neuronal Activity (A)

(B) Water

Lipids

10

8

6

4

2

0

PPM (C)

(D)

× 50 10

8

6

4

2

0

PPM

Figure 3.4. Effects of spatial localization. (A) Anatomical MR image of rat brain in vivo and (B) the corresponding proton NMR spectrum from the entire sensitive volume of the surface coil, which was placed on top of the rat. (D) Proton NMR spectrum from the localized volume as indicated in (C). Resonances from extracranial lipids are absent and the narrow water resonance line indicates the higher magnetic ﬁeld homogeneity of the volume of interest.

For an adequate tissue characterization it is essential to employ a more robust spatial localization technique such that the origin of the NMR signal is accurately known without contamination from other spatial positions. In the case of the brain as shown in Figure 3.4, this means that NMR signals from extracranial tissue should be eliminated and the localized volume should be placed in homogeneous brain structures, if possible. In most applications, spatial localization is achieved by applying a frequency-selective RF pulse in the presence of a magnetic ﬁeld gradient. The gradient makes the Larmor frequency linearly dependent on spatial position. Since the RF pulse only affects a selective range of frequencies, the combination results in the excitation (or refocusing/inversion) of a spatial slice. Three-dimensional localization is achieved by selecting the cubic intersection of three orthogonal slices. Figure 3.4(D) shows the 1 H NMR spectrum as acquired from a localized (cubic) volume as indicated on the image of Figure 3.4(C). The methyl and methylene protons from (extracranial) fats and lipids no longer contribute to the spectrum, leaving the water resonance and low-intensity resonances (not visible in Figure 3.4) from cerebral metabolites. Due to the decreased volume of interest, the main magnetic ﬁeld homogeneity is substantially improved, leading to narrower resonance lines and hence an improved spectral resolution. Single-volume spatial localization can be achieved with many different NMR sequences, of which ISIS (27), STEAM (28, 29) and PRESS (30) are most commonly used.

3.3.2. Shimming NMR spectroscopy applications place stringent requirements on the magnetic ﬁeld homogeneity. For example, most resonances observed with 1 H NMR spectroscopy resonate within a range of several ppm. In

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order to distinguish different chemical compounds it is therefore crucial to reduce the magnetic ﬁeld inhomogeneities to a small fraction of this chemical shift dispersion. Even though spatial localization inherently improves the magnetic ﬁeld homogeneity (see Section 3.3.1), this is often not sufﬁcient and the technique of shimming is applied to improve the magnetic ﬁeld homogeneity. Shimming is the process of canceling the magnetic ﬁeld inhomogeneities with additional spatially dependent (but exactly deﬁned) magnetic ﬁelds. Most NMR systems are equipped with shim coils that generate magnetic ﬁelds describing a particular order of spherical harmonics. Most NMR systems are equipped with at least eight shim coils, describing all ﬁrst and second order spherical harmonics that can be used to cancel magnetic ﬁeld inhomogeneities that vary linearly and quadratically with spatial position. Figure 3.5 shows a typical example of the spectral resolution and sensitivity enhancement achievable with shimming. The optimization of the magnetic ﬁeld homogeneity used to be an iterative, time-consuming and user-dependent process susceptible to local minima of

(C)

(B)

+ 40

Frequency (Hz)

(A)

– 40 (E)

40

Frequency (Hz)

Frequency (Hz)

(D)

0

40

0

−40

−40 −5

0

−5

5

0

Distance (mm) (F)

5

Distance (mm) (G) 7.0 Hz

12.8 Hz

−40

0 Frequency (Hz)

40

−40

0

40

Frequency (Hz)

Figure 3.5. Effects of optimizing the magnetic ﬁeld homogeneity. (A) Anatomical MR image of rat brain in vivo, indicating the rectangular volume of interest (VOI). (B, C) Two-dimensional images (‘phase maps’), (D, E) one-dimensional traces of the magnetic ﬁeld homogeneity and (F, G) NMR spectra of water from the VOI before (B, D, F) and after (C, E, G) optimization of the magnetic ﬁeld homogeneity over the VOI with all ﬁrst- and second-order shims. The one-dimensional traces (D, E) were extracted from (B, C) in the middle and along the long axis of the VOI. Note that an improved homogeneity within the VOI often leads to a reduced homogeneity outside the VOI (e.g. compare the somatosensory cortex/corpus callosum and caudate putamen areas in (B) and (C)).

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Brain Energetics and Neuronal Activity

suboptimal shim settings. Currently, a variety of noniterative and user-independent phase mapping techniques exist that can determine the optimal shim settings quantitatively within minutes (31, 32).

3.3.3. Water Suppression One of the main problems encountered during in vivo 1 H MRS studies is the large dynamic range of the resonances. The water signal is typically ∼10+3 –10+4 times stronger than the metabolite resonances (Figures 3.6(A) and (B)). Although a 16-bit analog-to-digital (ADC) converter should be able to adequately digitize the low metabolite resonances, the large water signal may also distort the baseline and introduce spurious sidebands (Figure 3.6(B)). For most 1 H MRS applications the water signal needs to be suppressed, without disturbing the metabolite resonances. The suppression of a particular resonance in a NMR spectrum requires a difference in a property between the molecule of interest and the interfering compound (e.g. water). This property need not to be a magnetic one, but it must be reﬂected in an NMR observable parameter. Thus, besides differences in chemical shift, scalar coupling and T1 and T2 relaxation, properties such as diffusion and exchange can also be exploited. Although there is no universal technique, there are some criteria by which the existing water suppression methods can be evaluated. These criteria include (1) degree of suppression, (2) insensitivity to RF imperfections (inhomogeneities), (3) ease of phasing the spectra, (4) minimum attainable repetition and echo times, (5) insensitivity to relaxation effects, (6) perturbation of other resonances and (7) degree of distortion of resonances near or at the water frequency. (A) Water

(B) Water tCr Cho

NAA 75

(C) NAA

tCr Cho tCr

Glx × 10

8

7

6

5

4 3 PPM

2

1

0

Figure 3.6. Effects of water suppression. (A) Localized proton NMR spectrum from rat brain in vivo without water suppression and (C) with CHESS water suppression. (B) Identical to (A) with an expanded vertical scale.

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The existing water suppression techniques can be divided in four groups, namely (1) methods that employ frequency-selective excitation and/or refocusing (33–36) or (2) utilize differences in relaxation and diffusion parameters (37–39), (3) spectral editing methods such as polarization transfer (39) and (4) other methods, including software-based water peak elimination. The ﬁrst group of water suppression techniques is currently most popular and, as a prominent example, the method of CHEmical Shift Selective (CHESS) water suppression (33) will be brieﬂy discussed here. CHESS consists of a frequency-selective excitation pulse for the water followed by a magnetic ﬁeld gradient. Therefore, the water is excited onto the transverse plane, where it will be destroyed (dephased) by the magnetic ﬁeld gradient, while the metabolite resonances reside along the longitudinal axis. Next, the metabolites can be excited onto the transverse plane and be detected (Figure 3.6(C)). The CHESS pulse sequence element may be repeated a number of times to improve the water suppression. Several related techniques exist, which, for example, do not excite the water (e.g. binomial RF pulses (36)), do not refocus the water (e.g. MEGA (34)) or which saturate the water resonance (e.g. presaturation). CHESS, and all other water suppression techniques have to make compromises between the criteria mentioned above.

3.3.4. Signal Quantiﬁcation NMR is, in principle, a quantitative technique and as such NMR spectra can be used to derive absolute concentrations of metabolites in animal and human tissues. This originates from the fact that the total integrated area under a resonance in an NMR spectrum (or the ﬁrst data point of a FID) is proportional to the longitudinal thermal equilibrium magnetization vector, which in turn is directly related to the number of spins in the sample and hence to the concentration of that particular compound. However, in practice the derivation of absolute concentrations by MRS is not straightforward, since many additional factors can inﬂuence the metabolite resonance area. These factors can include T1 and T2 relaxation, diffusion, exchange, (partial) NMR invisibility, spectral overlap of resonances and the choice of an internal or external concentration reference. The literature on signal quantiﬁcation by NMR is extensive (e.g. see (19) for reviews), but can essentially be simpliﬁed to a three-step process. First, one has to ensure that the integrated resonance area is directly proportional to the longitudinal thermal equilibrium magnetization. In many cases this can be achieved by proper experimental design. For example, an NMR spectrum acquired with a short repetition time and a long echo time will be heavily T1 and T2 weighted and the resonance areas will therefore not be representative of the thermal equilibrium magnetization. For this particular example this can be avoided by performing the experiment with a long repetition time (equal to ﬁve times the longest T1 relaxation time) and a short echo time (ideally an echo time of zero). In other cases, one may be able to derive a correction factor, whereas in the case of (partial) NMR invisibility due to excessive line widths, the quantiﬁcation process will always underestimate the actual concentration. The second step is to determine the integrated resonance area. For in vivo MRS applications this is not a trivial exercise, since many resonances are (partially) overlapping, have a complex scalar coupling splitting pattern or are superimposed on an intense baseline. For these reasons, simple integration is normally not feasible and one has to resort to more complicated time- or frequency-domain ﬁtting algorithms. After many years of development, two methods appear to crystallize as best suited for in vivo NMR spectroscopy. VARPRO (40) and related techniques appear to be well suited for ﬁtting of relatively few resonances, such as those observed in 31 P NMR spectra. This time-domain algorithm is ﬂexible for varying chemical shifts and scalar couplings, can readily avoid problems associated with intense baselines and is now routinely used (41, 42). LCmodel (43–45) is becoming the method of choice for more complex resonances, such as those observed in short echo-time 1 H NMR spectra. The LCmodel algorithm approximates the in vivo NMR spectrum as a linear combination of in vitro model solution spectra. Since all resonances of a given compound, e.g. glutamate, are ﬁtted simultaneously the great complexity of the glutamate NMR spectrum actually helps the convergence. Figure 3.7 shows a typical example of resonance quantiﬁcation of a short

44

Brain Energetics and Neuronal Activity (A)

(B) (C)

(D) (E) Glu Glu + Gln

Gln

Glu + Gln Lac Ala

(F) NAA

Cr + PCr Cr PCr mI

4.0

mI

mI

NAA

3.0

2.0

1.0

PPM

Figure 3.7. Spectral quantiﬁcation of proton NMR spectra with LCmodel. (A) Experimental proton NMR spectra acquired from rat brain in vivo (100 ml, STEAM localization, TR = 4000 ms, TE = 8 ms, TM = 25 ms, NEX = 128) overlaid with the ﬁtted NMR spectrum calculated by the LCmodel algorithm. (B) Difference between the calculated and measured spectra in (A). (C) Experimentally measured macromolecule baseline (double inversion recovery with TR = 6000 ms, TI1 = 1950 ms, TI2 = 550 ms) as included in the LCmodel algorithm. (D) Additional cubic spline baseline required for an optimal spectral ﬁt. Note that the macromolecule spectrum in (C) accounts for the majority of spectral baseline. (E, F) NMR spectra of individual compounds as extracted from the LCmodel ﬁt shown in (A) for (E) alanine (Ala), glutamate (Glu), glutamine (Gln) and lactate (Lac) and (F) myo-inositol (mI), total creatine (sum of creatine (Cr) and phosphocreatine (PCr)) and N-acetyl aspartate (NAA).

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echo-time 1 H NMR spectrum by the LCmodel algorithm. The ﬁnal step in the quantiﬁcation process is the conversion of relative resonance areas into absolute metabolite concentrations. This is normally done by comparing the metabolite resonance area with that of an internal or external reference compound of known concentration. The advantage of an external concentration reference is that the concentration is exactly known. However, this approach will introduce uncertainties about differences in coil loading and coil sensitivity between the metabolites and the concentration reference. The advantages of an internal concentration reference are exactly opposite, i.e. exact concentration must be assumed, but the metabolite and reference compound are acquired in an identical manner. In most recent applications, an internal concentration reference is used. Total creatine (sum of creatine and phosphorylated creatine) or water are commonly used.

3.4. ADVANCED EXPERIMENTAL TECHNIQUES 3.4.1. Introduction to Spectral Editing A signiﬁcant number of compounds cannot be unambiguously detected by (direct) in vivo 1 H NMR spectroscopy due to severe spectral overlap of resonances. The most commonly encountered examples of overlapping resonances in vivo are, lactate (and alanine) and lipids at ∼1.3 ppm, glutamate, glutamine, aspartate, N -acetyl aspartate and GABA between 1.9 and 2.4 ppm, GABA and total creatine at ∼3.0 ppm, and glucose and a wide range of other resonances between 3 and 4 ppm. There are several options to eliminate the overlapping resonances in order to unambiguously detect the metabolite of interest. Spectral editing techniques are the most straightforward methods to achieve this goal in vivo. Spectral editing may include all techniques that can simplify an NMR spectrum in order to limit the detection to speciﬁc metabolites. According to this general deﬁnition, spectral editing includes water suppression, spatial localization, use of shift reagents, selective excitation/refocusing and techniques utilizing differences in T1 and T2 relaxation, diffusion and magnetization transfer characteristics. However, to follow conventions of the (in vivo) NMR community, spectral editing is often restricted to those techniques which utilize the scalar coupling between spins to discriminate coupled from uncoupled spins. Two-dimensional (correlation) NMR spectroscopy can then be seen as the ultimate spectral editing technique, making no assumptions about the spin systems under investigations. The (one-dimensional) spectral editing techniques are generally more sensitive and less susceptible to motion, but require assumptions in order to selectively detect a given spin system. 3.4.1.1. Intermezzo : Scalar coupling A rigorous description of NMR requires the involvement of quantum mechanics and this is especially true for scalar-coupled spin systems. However, the interested reader is referred to excellent textbooks dealing with the quantum mechanics of NMR (46, 47). Here we will proceed with more intuitive and qualitative arguments to explain scalar coupling and formulate a number of practical rules that will allow one to understand the principle of the experiments described in the next section. At ﬁrst sight an NMR spectrum is completely characterized by the resonance frequencies and relative amplitudes originating from the nuclei under investigation. However, closer inspection reveals that most resonances are split into several smaller lines. This phenomenon of spin–spin or scalar coupling originates from the fact that nuclei with magnetic moments can inﬂuence each other, not only directly through space (dipolar coupling) but also through electrons in chemical bonds (scalar coupling). Even though dipolar interactions are the main mechanism for relaxation in a liquid, there is no net interaction between nuclei since rapid molecular tumbling averages the dipolar interactions to zero. However, interactions through chemical bonds do not average to zero and give rise to the phenomenon of scalar (or J ) coupling.

46

Brain Energetics and Neuronal Activity

In order to understand scalar coupling, consider the interaction between a proton and a carbon-13 nucleus (e.g. in [1-13 C]-glucose). For the proton there are two distinct states in which the magnetic moment of the scalar coupled 13 C nucleus is either parallel or anti-parallel to the external magnetic ﬁeld. The proton ‘senses’ these different states which are conveyed through the electron spin in the covalent 1 H– 13 C chemical bond. As a result the energy levels of the proton will degenerate, leading to two frequencies separated by the scalar coupling constant J . The same situation arises for the 13 C nucleus that senses two different orientations for the proton and also splits into two resonances at slightly different resonances. When there are more then two nuclei involved (as in a H3 13 C methyl group) the resonances split in a binomial pattern. For example, the 13 C nucleus splits into two resonances due to the ﬁrst proton. Each resonance splits again due to the second proton and again due to the third proton, ﬁnally appearing as four resonances, each separated by the scalar coupling constant at relative intensities of 1:3:3:1 (a so-called quartet). The protons only experience a single 13 C nucleus and therefore only split once into a doublet of relative intensity 1:1. Note that equivalent protons do not split among themselves due to magnetic equivalence rules. Further note that these splitting rules are only valid in the weak coupling approximation when the frequency difference between the resonances (e.g. 1 H and 13 C) is much larger than the scalar coupling constant. All heteronuclear scalar couplings are weak couplings, while most of the homonuclear scalar couplings (especially in proton NMR) are in the strong coupling limit. A noticeable exception is lactic acid, which is represented by a doublet resonance at 1.31 ppm and a quartet resonance at 4.10 ppm, even at low magnetic ﬁeld strengths.

3.4.2. Homonuclear Spectral Editing Homonuclear spectral editing is aimed at spectral simpliﬁcation by utilizing the homonuclear (commonly the proton–proton) scalar coupling to selectively detect or eliminate NMR resonances. The scalar coupling is utilized in a so-called Hahn spin-echo sequence, which is characterized by six distinct periods/operations (Figure 3.8(A)). Following a recovery delay (TR) in which the longitudinal magnetization is restored after any previous perturbations, the magnetization is excited onto the transverse plane by a 90◦ excitation pulse. During a time period TE/2, the transverse magnetization evolves under the inﬂuence of chemical shift, magnetic ﬁeld inhomogeneities, T2 relaxation and scalar coupling. The 180◦ refocusing pulse inverts the acquired phase evolution due to chemical shift and magnetic ﬁeld inhomogeneities, which will be perfectly refocused after the second TE/2 delay at the start of the signal detection period. However, the effects of T2 relaxation and scalar coupling are not refocused by the 180◦ pulse and lead to echo-time (TE) dependent signal loss and phase evolution, respectively. A complete description of the phase evolution due to scalar coupling (‘J evolution’) is beyond the scope of this chapter, but Figures 3.8(B) and (C) summarize the evolution of two important spin systems in vivo, namely lactic acid and γ -aminobutyric acid (GABA). It follows that the integrated transverse magnetization for the lactate methyl protons is described by a cosinusoidal modulation. At TE = 1/J the lactate doublet (1.31 ppm) will be inverted relative to uncoupled spins (like water), while at TE = 1/2J the resonance appears as an antiphase doublet, with zero integrated intensity. GABA-H4, which is scalar coupled to two protons of GABA-H3, appears as a triplet at 3.01 ppm and will therefore have a more complicated J -coupling modulation. The inner resonance of GABA-4 does not modulate as a function of TE and essentially behaves as an uncoupled spin. The outer resonances have a cosinusoidal modulation and are completely inverted at T E = 1/2J . Uncoupled spin systems do not have scalar coupling modulation and decrease exponentially with increasing echo time. This difference is utilized in spectral editing to differentiate between scalar-coupled and uncoupled spin systems. One additional tool is required to achieve homonuclear spectral editing, namely frequency-selective RF pulses. An RF pulse that selectively refocuses one spin in a scalar-coupled multi-spin system effectively inhibits J evolution, such that only T2 relaxation affects the signal intensity as a function of the echo time TE (Figure 3.8). The results in Figure 3.8 indicate that the simplest form of homonuclear spectral

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(A) 90°

TR

180°

TE/2

TE/2

(B)

(C)

Integrated Mxy

1

0

TE = 1/2J TE = 1/J

TE = 0

TE = 1/2J TE = 1/J

1/J 2/J Echo-time TE (E)

(D) Integrated Mxy

TE = 0 0

1

0 0

1/J

2/J

Echo-time TE

Figure 3.8. Effects of scalar coupling. (A) Generalized spin-echo sequence with a total echo time of TE. (B) Integrated transverse magnetization for noncoupled spins (exponential) and a scalar-coupled two-spin system (cosinusoidal). (C) Spectral appearance for a scalar-coupled two-spin system at echo times TE of 0, 1/2J and 1/J . Note that the spectrum at TE = 1/2J is phase corrected by 90◦ relative to the other spectra. (D) Integrated transverse magnetization for noncoupled spins (exponential) and the A spin in a scalar-coupled three-spin AX2 system (e.g. GABA-H4 and H2). (E) Spectral appearance at echo times TE of 0, 1/2J and 1/J . When performing a selective spin-echo, the integrated transverse magnetization for a scalar-coupled spin system will be identical to noncoupled spins.

editing, namely J difference editing (48, 49), comes down to the acquisition of two experiments. In the ﬁrst experiment a nonselective spin-echo is employed, leading to J evolution of the spin system of interest. At a total TE of 1/2J , the outer resonances of GABA-H2 and GABA-H4 are inverted. In the second experiment a frequency-selective spin-echo is executed, with a refocusing pulse that is selective for GABA-H2 and GABA-H4 (but not for GABA-H3). As a result, the J evolution of GABA is inhibited leading to positive resonances for GABA-H2 and GABA-H4. Spectral editing is achieved by subtracting the two data sets from each other. The (uncoupled) resonances from other metabolites are refocused by either refocusing pulse and will therefore appear identical in both experiments. Subtraction of the two data sets will therefore lead to a cancellation of the unwanted resonances. The outer resonances of GABA-H2 and GABA-H4 modulate between the two experiments and will therefore survive the subtraction, forming the edited signal. The inner resonance of GABA-H2 and GABA-H4 behaves as an uncoupled spin system and is lost in the subtraction process. The theoretical implementation of homonuclear J difference editing depicted in Figure 3.8 has to be extended with spatial localization and water suppression techniques for applications in vivo. Figure 3.9

48

Brain Energetics and Neuronal Activity (A)

tCr Selective inversion of GABA-H3 tCr Cho NAA

(B) Selective inversion at control position

(C) GABA-H4

GABA-H2

(D) Glx

NAA 4.0

3.0

2.0

1.0

PPM

Figure 3.9. Homonuclear J difference spectral editing of GABA on rat brain in vivo. (A, B) Proton NMR spectra (160 ml, TR = 3000 ms, TE = 68 ms, NEX = 128) obtained (A) with selective refocusing of GABA-H3 (inhibiting J evolution) and (B) with selective refocusing at a control position. (C) Difference between (A) and (B), showing editing resonances from GABA-H4 and GABA-H2 as well as a co-edited resonance from total glutamate (sum of glutamate and glutamine) and an inverted resonance from NAA due to the ﬁnite bandwidth of the selective refocusing pulse. (D) Edited GABA spectrum acquired 2 h following inhibition of the GABA degradation pathway by vigabatrin. Note the splitting of the edited GABA-H4 resonance, indicating a minimal contribution from macromolecules.

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shows a typical in vivo result for GABA editing on rat brain. Besides J difference editing, homonuclear spectral editing includes a wide range of techniques, utilizing different manipulations of the scalar coupling and J evolution (see (19) for review). Each spectral editing technique has unique characteristics in terms of sensitivity, ease of use, motion, suppression of unwanted resonances and sensitivity to system imperfections. As a rough guideline, J difference editing is close to optimal for stationary objects such as the brain. For more demanding applications, other techniques may be more optimal.

3.4.3. Heteronuclear Spectral Editing In analogy to homonuclear spectral editing (Section 3.4.2), heteronuclear spectral editing is aimed at the simpliﬁcation of NMR spectra by selectively detecting NMR resonances based on the absence or presence of heteronuclear scalar coupling. The principle of heteronuclear J difference editing, or POCE (ProtonObserved, Carbon-Edited) NMR spectroscopy (50), is analogous to the homonuclear version with some practical exceptions. Since the heteronuclear J coupling is often much larger than homonuclear J coupling constants (J1H13C = 120–180 Hz versus J1H1H = 1–15 Hz), the optimal echo time 1/J is much shorter (normally TE ∼ 7–9 ms). Furthermore, since protons can form only a single covalent bond, heteronuclear scalar coupling always lead to the simplest splitting, i.e. a doublet. A doublet can be edited with 100 % (A) NAA tCr

tCr

Cho Glu

Glx Gln

Ins Tau

NAA Asp

Lac

(B) Glu-H4 Glx-H2 Glc-H6

Asp-H2

tCr GABA-H4

Gln-H4

Glx-H3 NAA Ala-H3

Asp-H3

Lac-H3 ×4

4.0

3.0

2.0

1.0

PPM

Figure 3.10. (A) Nonedited and (B) edited 1 H-[13 C] NMR spectra from rat brain in vivo as averaged from a 100 µl volume over 25 min between 85 and 110 min following the onset of intravenous [1,6-13 C2 ]-glucose infusion.

50

Brain Energetics and Neuronal Activity

efﬁciency, unlike other more complicated splitting patterns. Heteronuclear editing also requires selective RF pulses, but the large difference in resonance frequency between protons (300 MHz at 7 T) and for instance carbon-13 (75 MHz at 7 T) ensures that any RF pulse applied to one nucleus is automatically selective for the other nucleus. Besides some additional technical challenges, such as decoupling, heteronuclear editing is identical to homonuclear editing. Two experiments are required, with and without a (selective) RF pulse on the carbon-13 channel. Subtracting the two data sets give the proton NMR resonances from spins that are coupled to carbon-13 spins, while the sum gives the spins that are not scalar coupled (i.e. are bound to a carbon-12 nucleus). The fractional enrichment of any metabolite can be calculated directly from the ratio (1 H-[13 C]/1 H-[12 C +13 C]). The fractional enrichment, in combination with the absolute concentration, form the basis of metabolic modeling and interpretation of 13 C turnover data, as will be described in detail in several of the following chapters. Figure 3.10 shows a typical example of a 1 H-[13 C]-NMR spectrum obtained from rat brain between 60 and 80 min following the infusion of [1, 6-13 C2 ]-glucose.

REFERENCES 1. Purcell EM, Torrey HC, Pound RV, Resonance absorption by nuclear magnetic moments in a solid. Phys Rev 1946; 69: 37–38 2. Bloch F, Hansen WW, Packard ME, Nuclear induction. Phys Rev 1946; 69: 127 3. Bloch F, Nuclear induction. Phys Rev 1946; 70: 460–473 4. Bloch F, Hansen WW, Packard ME, The nuclear induction experiment. Phys Rev 1946; 70: 474–485 5. Proctor WG, Yu FC, The dependence of a nuclear magnetic resonance frequency upon chemical compound. Phys Rev 1950; 77 6. Dickinson WC, Dependence of the F19 nuclear resonance position on chemical compound. Phys Rev 1950; 77: 736 7. Ernst RR, Anderson WA, Applications of Fourier transform spectroscopy to magnetic resonance. Rev Sci Instrum 1966; 37: 93–102 8. Shaw TM, Elsken RH, Nuclear magnetic resonance absorption in hygroscopic materials. J Chem Phys 1950; 18: 1113–1114 9. Odebald E, Lindstrom G, Some preliminary observations on the proton magnetic resonance in biological samples. Acta Radiol 1955; 43: 469–476 10. Damadian R, Tumor detection by nuclear magnetic resonance. Science 1971; 171: 1151–1153 11. Moon RB, Richards JH, Determination of intracellular pH by 31P magnetic resonance. J Biol Chem 1973; 248: 7276–7278 12. Hoult DI, Busby SJ, Gadian DG, Radda GK, Richards RE, Seeley PJ, Observation of tissue metabolites using 31P nuclear magnetic resonance. Nature 1974; 252: 285–7 13. Chance B, Nakase Y, Bond M, Leigh JS, Jr., McDonald G, Detection of 31 P nuclear magnetic resonance signals in brain by in vivo and freeze-trapped assays. Proc Natl Acad Sci U S A 1978; 75: 4925–9 14. Ackerman JJ, Grove TH, Wong GG, Gadian DG, Radda GK, Mapping of metabolites in whole animals by 31P NMR using surface coils. Nature 1980; 283: 167–70 15. Behar KL, den Hollander JA, Stromski ME, Ogino T, Shulman RG, Petroff OA, Prichard JW, High-resolution 1H nuclear magnetic resonance study of cerebral hypoxia in vivo. Proc Natl Acad Sci U S A 1983; 80: 4945–8 16. Alger JR, Sillerud LO, Behar KL, Gillies RJ, Shulman RG, Gordon RE, Shae D, Hanley PE, In vivo carbon-13 nuclear magnetic resonance studies of mammals. Science 1981; 214: 660–2 17. Lauterbur PC, Image formation by induced local interactions: examples employing nuclear magnetic resonance. Nature 1973; 242: 190–191 18. Mansﬁeld P, Multiplanar image formation using NMR spin echoes. J Phys C: Solid State Phys 1977; 10: L55–L58 19. de Graaf RA, In vivo NMR Spectroscopy. Principles and Techniques. 1998, Chichester: Wiley. 20. Behar KL, Ogino T, Characterization of macromolecule resonances in the 1H NMR spectrum of rat brain. Magn Reson Med 1993; 30: 38–44

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21. Behar KL, Rothman DL, Spencer DD, Petroff OA, Analysis of macromolecule resonances in 1H NMR spectra of human brain. Magn Reson Med 1994; 32: 294–302 22. Govindaraju V, Young K, Maudsley AA, Proton NMR chemical shifts and coupling constants for brain metabolites. NMR Biomed 2000; 13: 129–53 23. Ugurbil K, Magnetization transfer measurements of individual rate constants in the presence of multiple reactions. J Magn Reson 1985; 64: 207–219 24. Cohen SM, Ogawa S, Shulman RG, 13C NMR studies of gluconeogenesis in rat liver cells: utilization of labeled glycerol by cells from euthyroid and hyperthyroid rats. Proc Natl Acad Sci U S A 1979; 76: 1603–9 25. Stromski ME, Arias-Mendoza F, Alger JR, Shulman RG, Hepatic gluconeogenesis from alanine: 13C nuclear magnetic resonance methodology for in vivo studies. Magn Reson Med 1986; 3: 24–32 26. Sillerud LO, Shulman RG, Structure and metabolism of mammalian liver glycogen monitored by carbon-13 nuclear magnetic resonance. Biochemistry 1983; 22: 1087–94 27. Ordidge RJ, Connelly A, Lohman JA, Image-selected in vivo spectroscopy (ISIS). A new technique for spatially selective NMR spectroscopy. J Magn Reson 1986; 66: 283–294 28. Frahm J, Merboldt KD, Hanicke W, Localized proton spectroscopy using stimulated echoes. J Magn Reson 1987; 72: 502–508 29. Granot J, Selected volume excitation using stimulated echoes (VEST). Application to spatially localized spectroscopy and imaging. J Magn Reson 1986; 70: 488–492 30. Bottomley PA, Spatial localization in NMR spectroscopy in vivo. Ann N Y Acad Sci 1987; 508: 333–348 31. Shen J, Rothman DL, Hetherington HP, Pan JW, Linear projection method for automatic slice shimming. Magn Reson Med 1999; 42: 1082–8 32. Gruetter R, Automatic, localized in vivo adjustment of all ﬁrst- and second-order shim coils. Magn Reson Med 1993; 29: 804–11 33. Haase A, Frahm J, Hanicke W, Matthaei D, 1H NMR chemical shift selective (CHESS) imaging. Phys Med Biol 1985; 30: 341–344 34. Mescher M, Tannus A, O’Neil Johnson M, Garwood M, Solvent suppression using selective echo dephasing. J Magn Reson A 1996; 123: 226–229 35. de Graaf RA, Nicolay K, Adiabatic water suppression using frequency selective excitation. Magn Reson Med 1998; 40: 690–6 36. Hore PJ, Solvent suppression in Fourier transform nuclear magnetic resonance. J Magn Reson 1983; 55: 283–300 37. Patt SL, Sykes BD, Water eliminated Fourier transform NMR spectroscopy. J Chem Phys 1972; 56: 3182–3184 38. van Zijl PCM, Moonen CTW, Complete water suppression for solutions of large molecules based on diffusional differences between solute and solvent (DRYCLEAN). J Magn Reson 1990; 87: 18–25 39. van Zijl PCM, Moonen CTW, Solvent suppression for in vivo magnetic resonance spectroscopy. In NMR Basic Principles and Progress, Diehl, P, Fluck, E, Gunther, H, Kosfeld, RSeelig, J (Eds). 1992, Berlin: Springer-Verlag, pp. 67–108. 40. van der Veen JW, de Beer R, Luyten PR, van Ormondt D, Accurate quantiﬁcation of in vivo 31 P NMR signals using the variable projection method and prior knowledge. Magn Reson Med 1988; 6: 92–8 41. Maintz D, Heindel W, Kugel H, Jaeger R, Lackner KJ, Phosphorus-31 MR spectroscopy of normal adult human brain and brain tumours. NMR Biomed 2002; 15: 18–27 42. van den Boogaart A, Howe FA, Rodrigues LM, Stubbs M, Grifﬁths JR, In vivo 31 P MRS: absolute concentrations, signal-to-noise and prior knowledge. NMR Biomed 1995; 8: 87–93 43. de Graaf AA, Bovee WM, Improved quantiﬁcation of in vivo 1 H NMR spectra by optimization of signal acquisition and processing and by incorporation of prior knowledge into the spectral ﬁtting. Magn Reson Med 1990; 15: 305–19 44. Provencher SW, Estimation of metabolite concentrations from localized in vivo proton NMR spectra. Magn Reson Med 1993; 30: 672–9 45. Pfeuffer J, Tkac I, Provencher SW, Gruetter R, Toward an in vivo neurochemical proﬁle: quantiﬁcation of 18 metabolites in short-echo-time (1)H NMR spectra of the rat brain. J Magn Reson 1999; 141: 104–20 46. Slichter CP, Principles of Magnetic Resonance. 1990, Berlin: Springer-Verlag. 47. Ernst RR, Bodenhausen G, Wokaun A, Principles of Nuclear Magnetic Resonance in One and Two Dimensions. 1987, Oxford: Clarendon Press.

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48. Rothman DL, Behar KL, Hetherington HP, Shulman RG, Homonuclear 1 H double-resonance difference spectroscopy of the rat brain in vivo. Proc Natl Acad Sci U S A 1984; 81: 6330–4 49. Rothman DL, Petroff OA, Behar KL, Mattson RH, Localized 1 H NMR measurements of gamma-aminobutyric acid in human brain in vivo. Proc Natl Acad Sci U S A 1993; 90: 5662–6 50. Rothman DL, Behar KL, Hetherington HP, den Hollander JA, Bendall MR, Petroff OA, Shulman RG, 1 HObserve/13 C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo. Proc Natl Acad Sci U S A 1985; 82: 1633–7

4 Metabolic Modeling Analysis of Brain Metabolism Graeme Mason Department of Psychiatry, Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

4.1 Introduction 4.2 Basic Elements of a Metabolic Model for 13 C NMR Studies 4.2.1 Metabolic Pools 4.2.2 Sources of Substrates Supplied to the System 4.2.3 Rates of Metabolic Pathways 4.2.4 Combination Pools 4.2.5 Targets 4.3 Uniting the Elements to Create a Model 4.3.1 Mass and Isotope Balance Equations 4.3.2 Constraints upon a Model 4.3.3 Simpliﬁcations of a Model 4.3.4 Isotopomer Analysis in a Model 4.4 Mechanics of Fitting Data 4.4.1 Least-squares Fitting 4.4.2 Numerical Fitting Algorithms 4.4.3 Effects of Scatter 4.4.4 Information Content in a 13 C-Labeling Experiment 4.5 Conclusion

Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

54 57 57 59 59 59 60 60 60 61 61 61 62 62 63 64 66 69

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Brain Energetics and Neuronal Activity

4.1. INTRODUCTION Mathematical modeling of brain metabolism to interpret isotopic labeling data was pioneered by van den Berg and Garﬁnkel, who developed multi-compartmental models of brain metabolism that explained what were apparent paradoxes of 14 C biochemical labeling studies performed with radioisotopes (Van den Berg and Garﬁnkel 1971; Van den Berg et al., 1970). A limitation of the 14 C methodology is that it requires sacriﬁcing animals and chemical extraction of the brain to obtain data. Today, NMR spectroscopy has allowed isotopic labeling measurements of 13 C and other nuclei to be performed in vivo (Beckmann et al., 1991; Behar et al., 1986; Bluml et al., 2001a; Bluml et al., 2001b; Fitzpatrick et al., 1990; Gruetter et al., 1994; Gruetter et al., 2001; K¨unnecke et al., 1993; Moreno et al., 2001a; Moreno et al., 2001b; Rothman et al., 1992), in vitro (Sillerud et al., 1981; Sonnewald et al., 1991; Sonnewald et al., 1996; Szczepaniak et al., 1996; Waagepetersen et al., 2000), in perfused organs (Lewandowski et al., 1997; Yu et al., 1995), and in brain slices (Badar-Goffer et al., 1990; Badar-Goffer et al., 1992). In vivo MRS is less sensitive than 14 C methods, but has the advantages of allowing many more time points in turnover curves and can be applied to human studies. The major fuel for brain metabolism is glucose with monocarboxylic acids such as acetate, βhydroxybutyrate, and lactate acting as minor but signiﬁcant fuel sources. As described in detail in Chapters 5 and 6 there are two major cell types in the brain, neurons and glia. Neurons transmit electrical impulses and communicate with each other by release of chemicals called neurotransmitters. Glia surround the neurons and maintain a stable extracellular environment as well as providing the neurons with key substrates. There are many types of neurons, usually classiﬁed based upon morphology and the primary neurotransmitter they release. In the cerebral cortex over 90 % of the neurons and synapses are from glutamatergic and GABAergic neurons (Shephard, 1994). Although multi-compartment models are vulnerable to multiple solutions, in the brain the modeling is simpliﬁed by the presence of key synthetic enzymes and metabolite pools in speciﬁc cell types. For example glutamine is only synthesized in glial cells, GABA is only synthesized in GABAergic neurons, and brain glutamate is primarily in glutamatergic neurons. To a ﬁrst order these metabolite pools may be considered as reﬂecting cell speciﬁc metabolism, and each of the pools modeled separately. More sophisticated analysis allows the metabolic interactions between these cell types to be calculated (Rothman et al., 2003). A further simpliﬁcation in the modeling is that, to a resolution on the order of 200 µm, most of the cerebral cortex has its own arterial blood supply (see Chapter 8). Therefore, across the cortex, there is not the gradation of plasma substrate levels that complicates the modeling of hepatic and muscle metabolism. In this review we focus on the modeling of neuronal glucose metabolism, in particular the neuronal TCA cycle and, secondarily, the glutamine cycle. As described extensively in other chapters, the glutamate/glutamine cycle is a metabolic pathway shared between neurons and glia. In this pathway glutamate released by the neuron as a neurotransmitter is taken up by surrounding glial cells and converted to glutamine. Glutamine is not neuroactive and is released by the glia for uptake by the neurons to replace lost glutamate. Figure 4.1 shows the metabolic interactions of glutamate metabolism between glia and neurons. Measurement of the glutamate/glutamine cycle is of particular interest because it is a direct measurement of glutamate neurotransmission, which is the major excitatory neurotransmitter pathway in the cerebral cortex. Neuronal glucose oxidation by the TCA cycle is the main source of energy for the brain, and impairment of the TCA cycle is a symptom of several neurological and psychiatric diseases. In vivo NMR has the unique ability to measure both of these critical pathways simultaneously and noninvasively. In this chapter we focus on the principles for using mathematical modeling of dynamic and steadystate isotopic labeling to derive rates of brain metabolism, and some of the issues which are unique to modeling MRS data. The intention is primarily pedagogical, and readers are referred to other chapters in this book for current thinking on brain metabolism. Because glucose is the most common substrate used experimentally, we focus on the use of [1-13 C]glucose as an isotopic precursor. Figure 4.2 shows a

Metabolic Modeling Analysis of Brain Metabolism

55

Astrocyte

Neuron

Glutamine GS Glutamate

Glutamine PAG Glutamate

Cycle Cycle

TCAastrocyte

TCAneuron

Lactate

Glucose

Lactate

Figure 4.1. Schematic representation of the glutamate/glutamine cycle between neurons and glia. The diagram shows a two-compartment model of brain metabolism between glutamate-releasing (glutamatergic) neurons and surrounding astrocyte cells. Glutamate is released from the neuron through the dumping of the contents of synaptic vesicles upon depolarization. Released neurotransmitter glutamate is transported from the synaptic cleft by surrounding glial end processes, in a process that is driven by the glial sodium gradients. Once in the glia glutamate is converted to glutamine by glutamine synthetase (GS). Glutamine is released by the astrocyte, transported into the neuron, and converted to glutamate by phosphate-activated glutaminase (PAG), which completes the cycle. Glu C4,3 Glu C4 Gln C4

Glu C4,3 +GABA C2

Glu C3,4

[1-13C]Glucose infused Gln C2

Glucose C1 b

Glu C3

Glu C2

Glu C3,4 +Gln C3 NAA C6

NAA C2

Glucose C1 a

Asp C3 Asp C2

Lipid

Natural abundance

105

95

85

75

65

55 ppm

45

35

25

15

5

Figure 4.2. 13 C MRS spectrum in the occipital/parietal lobe at 2.1 T. The ﬁgure shows a 45 min accumulation 13 C MRS spectrum obtained at 2.1 T approximately 60 min after the start of a 1-13 C glucose infusion. The spectrum was obtained from a 144 ml volume centered on the midline in the occipital/parietal lobe. Labeled resonances include the C2, C3, and C4 positions of glutamate, glutamine, aspartate, and GABA and the C3 position of lactate. The localization of the synthetic enzymes and pools of GABA and glutamine to GABAergic neurons and glia, respectively, and the localization of the majority of the glutamate pool to glutamatergic neurons, allows the relative rates of glucose oxidation in these cell types to be determined from the ﬂow of 13 C label into these pools (Rothman et al., 2003). Figure from Mason et al., 2003.

56

Brain Energetics and Neuronal Activity Km, Vmax Go

Gi CMRgl G6P (0.211)

Vgly Vpp

GAP (0.065) Vgly L (1.5)

Vlout

Vtca Vana

Lo

Vdil

Cit4 (0.296) Vtca KG4 (0.220)

Vtcanet

Unlabeled sources

Vgln Vxnet Vx Vxa

OAA2 or 3

Glu4 (12) Asp

Gln4 (6.2)

Vefflux

Vcycle

Vtca Vtcanet

Cit3 Vtca KG3 (0.220)

Vgln Vxnet Vx

Glu3 (12)

Gln3 (6.2)

Vefflux

Vcycle

Figure 4.3. Schematic representation of ﬂow of 13 C from glucose through glutamate to glutamine in a simpliﬁed one-compartment model. This model is the initial model used to ﬁt dynamic time courses of brain isotopic labeling data. It is simpliﬁed from the neuronal/glial interactions shown in Figure 4.1 by not modeling internal glial metabolism. Provided that the amount of glial glutamate is small, this model provides an accurate description of neuronal metabolism when glutamine labeling, which is the main input to the neuron from the glial cells, is measured. The concentrations of each metabolite are given in parentheses next to the pool name, in units of mmol/kg. Glucose in the plasma (Go ) and the brain (Gi ) are in exchange at rates Vin and Vout ; these rates vary with plasma glucose concentration but can be predicted with Michaelis–Menten kinetics. The 13 C label ﬂows at the rate CMRgl through glucose-6-phosphate and fructose-6-phosphate, which are assumed to be in isotopic equilibrium and are therefore added to form a single pool (G6P). The pentose phosphate pathway removes label from G6P at the rate Vpp [0.03Vgly ; (Hawkins et al., 1985; Mason et al., 1992b)]. The 13 C label continues through glycolysis, passing through all of the intermediates from fructose-1,6-bisphosphate to phosphoenolpyruvate; these intermediates are assumed to be in isotopic equilibrium and are therefore added to form a single pool (GAP). The 13 C arrives at pyruvate and through exchange labels lactate; lactate and pyruvate are assumed to be in isotopic equilibrium and are therefore added to form a single pool (L). This pool is in exchange with plasma pyruvate and lactate (Lo ) at the rates Vlin [0.05 mmol/kg/min (Pardridge and Oldendorf, 1977)] and Vlout [0.085 mmol/kg/min; calculated from Pardridge and Oldendorf (1977) and intracerebral lactate concentrations reported by Hawkins and Mans (1983)]. The 13 C enters the TCA cycle and labels citrate and isocitrate at carbon 4; these pools are assumed to be in isotopic equilibrium and are added to form a single pool (Cit4 ). At α-KG and glutamate C4, the 13 C label exchanges between the mitochondrial and cytosolic pools of each; these exchanges are reduced to a single exchange between two grouped pools (KG4 and Glu4 ). Glutamate is released from the neuron, taken into the astrocyte, and converted to glutamine at the rate Vgln [0.08–0.5 mmol/kg/min (Sibson et al., 1998) and calculated from Dzubow and Garﬁnkel (1970)], labeling the C4 of glutamine (Gln4 ). The 13 C label continues through the TCA cycle to oxaloacetate, which then exchanges with aspartate (Asp); the TCA cycle intermediates between α-KG and glutamate C4 are assumed to be in isotopic equilibrium and are added to form a single pool (OAA). The 13 C label enters its second and all subsequent turns of the cycle at the lumped citrate pool (Cit3 ), so called because the 13 C now ﬂows to the C3 of α-KG (KG3 ), glutamate (Glu3 ), and glutamine (Gln3 ). 13 C ﬂow diverges at L at the pyruvate carboxylase rate Vpc [0.10 Vtca (Berl et al., 1962)]. Vefﬂux is equal to Vpc , which maintains the balance of mass in the TCA cycle. This ﬁgure was adapted from Mason et al. (1992b). Reproduced by permission of Lippincott, Williams and Wilkins.

Metabolic Modeling Analysis of Brain Metabolism

57

13

C NMR spectrum obtained from the human brain after intravenous infusion of [1-13 C]glucose. The key metabolites involved in glutamate metabolism, which are glutamate, glutamine, and γ -aminobutyric acid (GABA), are heavily labeled. By ﬁtting the in vivo labeling curves a with multi-compartment metabolic model, key metabolic rates can be calculated, including the rate of neuronal glucose metabolism, and the glutamate/glutamine cycle. Figure 4.3 shows a schematic diagram of a model of 13 C labeling from [1-13 C]glucose based on Figure 4.1, which will be referred to in the rest of this review. The caption to the ﬁgure describes the model in detail. This model is focused on neuronal metabolism in glutamatergic cells. However, it takes into account neuronal/glial interactions by treating glial glutamine labeling as an input. Although simpliﬁed relative to the models that we and other groups are currently using, we will use this one-compartment model to explain the concepts and procedures involved in the modeling analysis. Extensions to two and three compartments are described in Chapters 5 and 6 and the following references (Gruetter et al., 2001; Mason et al., 2001; Rothman et al., 2003; Sibson et al., 2001).

4.2. BASIC ELEMENTS OF A METABOLIC MODEL FOR

13

C NMR STUDIES

A metabolic modeling analysis of isotopic turnover data requires several basic elements: metabolic pools, sources of substrates supplied to the system, rates of metabolic pathways, and for ﬁtting, one needs the target data that have been measured experimentally. Some models also require the calculation of combination pools, which will be deﬁned in this section of the chapter. With these ﬁve basic elements, it is possible to generate equations that express any scenario of mass and isotopic ﬂow among metabolic pools and adjust them to obtain the closest approximation to real life.

4.2.1. Metabolic Pools Metabolic pools are characterized by four components. They are the initial concentration, the position of isotopic labeling that results from a particular ﬂow or combination of ﬂows, the inﬂows, and the outﬂows. The initial concentration is required because it controls the percent enrichment of the pool. Although consumption of a molecule degrades the entire chemical at the same rate through the same pathways, the carbon that is supplied to a molecule can be derived from different sources and paths. For example, in Figure 4.3, glutamate is used to synthesize glutamine or α-ketoglutarate. However, an examination of the individual atoms of glutamate shows that the C4 must come from carbon derived from acetyl-CoA, and the C3 may come from anaplerotic fueling of the C2 of oxaloacetate, or from carbon that originated in the C3 position of glutamate itself. In fact, each of the carbon positions of glutamate has a different set of sources. So, the C4 of glutamate and each of the other glutamate carbon positions function as distinct metabolic pools that happen to have the same rates of degradation by virtue of their residing in the same chemical but usually are not labeled by the same pathways. Investigators may also analyze the combinations of 13 C-labeling that can occur at multiple carbon positions within a molecule, and each of those combinations can be modeled as additional, distinct metabolic pools (Figure 4.3). The inﬂows and outﬂows are simply rates of transfer of mass and isotope into and out of the metabolic pool. The ﬂows can be of many types, including absolute rates, mass-dependent time constants of ﬂow, source-dependent ﬂows that are governed by Michaelis–Menten kinetics, or ﬂows that introduce feedback or feedforward regulation. Whatever the type of ﬂow, there come into play two key concepts, and which are turnover and accumulation (Figure 4.4), and carefully structured metabolic modeling becomes extremely important in the case of turnover. When 13 C arrives in a metabolite pool and replaces carbon that is leaving the pool, turnover has occurred. If the metabolite pool becomes labeled without an increase in concentration, then turnover has occurred. The

58

Brain Energetics and Neuronal Activity Vin

Vout

S 12

100

Vin = Vout

10 8

P

75

Total P

6

Vin = Vout

50

Labeled P 4

25

2

0

Vin > Vout 60

Percent enrichment

Concentration (mmol/kg)

0

Total P 40 Labeled P

20 0

75

Vin > Vout

50 25 0

Vin < Vout

10

75

8

Vin < Vout

Total P 50

6 4

25 Labeled P

2

0

0 0

20

40

60

Time (min)

0

20

40

60

Time (min)

Figure 4.4. The effects of ﬂow from a labeled substrate S into a product P at the respective rates Vin and Vout , for P = 10 mmol/kg, Vin = 1 mmol/kg/ min, and S is 100 % enriched. Top pair: if Vin = Vout , the total amount of P is constant, and the amount of labeled P increases as a single-exponential function, as does the percent enrichment of P . Middle pair: if Vin > Vout , the total amount of P and labeled P increase linearly. In the example here, Vout = 0. The percent enrichment can no longer be described by a single-exponential function. Bottom pair: if Vin < Vout , the total amount of P decreases linearly. In this example, Vout = 1.14 mmol/kg/ min. The amount of labeled P increases as Vin supplies label and increases the percent enrichment of P , but as the total quantity of P decreases, so eventually must the quantity of labeled P . It is interesting to see that the percent enrichment actually rises slightly more quickly for Vin < Vout , and this is because the amount of P to enrich decreases, requiring less and less labeled substrate to reach 100 % enrichment.

metabolite pool can decrease in level, still with the labeling described as turnover, but when the introduction of label occurs with an increase in total mass of the metabolite pool, the result is mass accumulation. Mass accumulation requires that the breakdown of the metabolite be slower than its synthesis. Referring to Figure 4.4, in a case of pure mass accumulation, the appearance of 13 C label represents pure synthesis, and the mass and isotope balance equations would be d[P]/dt = Vin

Metabolic Modeling Analysis of Brain Metabolism and

59

d[P∗ ]dt = ([S∗ ]/[S])Vin

One such case would be if the breakdown of lactate were completely blocked, and the rate of increase in 13 C-lactate were measured. In that case of pure accumulation, the slope of the time-dependent increase in 13 C-labeling of the product could be used directly for a simple, easy determination of its rate of synthesis (middle set of graphs of Figure 4.4). In contrast, turnover requires more intense mathematics to interpret than does mass accumulation. While accumulation means that mass is being added to the observed pool, turnover means that the sample is being changed rather than simply increased, with at least some of the sample leaving and being replaced by new material. That requires some breakdown of the product. Pure turnover occurs for Vin ≤ Vout . The determination of the metabolic rates under conditions of turnover is considerably more complicated than is the case for mass accumulation. The top and bottom panels of Figure 4.4 show the time courses of 13 Clabel accumulation and 13 C percent enrichment for when turnover occurs under conditions of Vin = Vout and under conditions of net loss of P (Vin < Vout ).

4.2.2. Sources of Substrates Supplied to the System Isotopic labeling measurements are based on an initial condition in which there is less of an isotope in the system than is present in the substrate that will be injected into the system. 13 C-labeling studies almost always begin with carbon having the 1.1 % of natural abundance, and that enrichment can be raised a little or a lot, depending on the experimental preparation and the pathways that are being evaluated. One common substrate, C1-labeled glucose, typically leads to labeling that is 15–30 % at the isotopic steady state in vivo, whereas C2-labeled glucose leads to labeling of only a few percent, because the pathways of entry are different. A labeled substrate should be characterized as a function of time, beginning with the condition before the start of the labeled infusion and continuing through the time that the last metabolic measurements are acquired. The time course of labeling is used as a driver for the system of equations that will be used to simulate metabolism. For example, the rate of labeling of brain glucose is the rate of ﬂow of 13 C-glucose from the blood to the brain, which is governed by blood–brain transport of glucose and by the availability of 13 C-glucose in the blood. Generally, the time courses of concentration and percent enrichment of any labeled substrates should be known for this measurement to be successful. In Figure 4.3, the C1 of glucose is labeled. It is important to remember that many substrates may contribute carbon to the chemicals whose labeling is observed in a study. For example, ketone bodies, acetate, pyruvate, and lactate all provide carbon that ﬂows to glutamate C4. During infusions of [1-13 C]glucose in vivo, the carbon from these alternative substrates remains unlabeled, but the substrates should still be included as sources of unlabeled carbon.

4.2.3. Rates of Metabolic Pathways For the neurochemicals to become labeled from the carbon substrates, the substrates and chemicals must be linked by rates of metabolic ﬂow. Although it is also possible in principle to adjust the sizes of metabolic compartments (Lebon et al., 2002), it is metabolic rates that are most often adjusted when ﬁtting experimental data. The rates of ﬂow can either be evaluated as absolute rates or as kinetic parameters that are used to calculate rates of ﬂow, such as Michaelis–Menten Km or Vmax (Mason et al., 1992a).

4.2.4. Combination Pools Although NMR spectroscopy is unrivaled in its chemical speciﬁcity, the speciﬁcity is not complete, especially for studies that are performed in vivo. Separate metabolic pools of the same chemical are often not

60

Brain Energetics and Neuronal Activity

distinguished, and sometimes even different chemicals cannot be resolved from one another, and for this purpose, most models that are analyzed in vivo require the use of combination pools. One example of the lack of differentiation of resonances from several metabolic pools is the compartmentation of glutamate among glutamatergic neurons, GABAergic neurons, and astroglia. Although distinct physical compartments exist for these pools of glutamate, there is currently no distinction among them as detected by NMR spectroscopy; what is observed is a combination pool. Therefore, to simulate observations of 13 C-labeling, metabolic models require the labeling of each pool to be calculated separately and then summed to compute the kinetics of the combination pool of total glutamate. Fortunately, the glutamate pools in the GABAergic and glial compartments are small relative to glutamatergic neurons, so the rates of metabolism calculated are relatively unaffected by uncertainties about their pool sizes (Lebon et al. 2002; Rothman et al. 2003).

4.2.5. Targets A simulation of metabolism by itself can be a useful tool to test the consequences of ideas, but the simulation should always be done in the context of actual data. So, any model should be able to be compared to experimental observations, which are called target data. A set of target data should include the time that corresponds to each NMR measurement and either a concentration or a percent enrichment of 13 C-labeling of a particular carbon position. If the concentration of the observed metabolic pool changes with time, then the target data should also include the total concentration (labeled plus unlabeled) material as a function of time. If 13 C-labeling is measured by tissue extraction, then the time of a particular data point in the target set is the time at which the tissue was removed and metabolism halted by freezing, microwaving, or chemical means. If 13 C-labeling is measured in vivo, the time of the data point should be the average of the time interval over which the signals were accumulated for the measurement. For example, in previously published studies of human brain metabolism, individual spectra were acquired every 5 min, so each time point corresponded to 2.5 min after the start of the acquisition.

4.3. UNITING THE ELEMENTS TO CREATE A MODEL The metabolic pools, substrate drivers, and rates of metabolic pathways are combined to create a set of mass balance equations and a set of isotope balance equations, with one of each for every relevant labeling pattern of each metabolic pool that is in the model. The mass and isotope balance equations can then be solved in light of various constraints that are placed on the model by mass balance and a priori knowledge from other experiments.

4.3.1. Mass and Isotope Balance Equations The mass balance equations are a mathematical statement of how the total mass of a metabolic pool changes over time. A mass balance equation has the general form dP = Vin − Vout dt

(4.1)

where dP /dt is the rate of change of the metabolic pool, Vin is the rate of ﬂow from the metabolite M, and Vout is the rate of ﬂow out of the pool (Figure 4.4). At a metabolic steady state, Vin = Vout , and the mass of the pool P does not change with time.

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61

The isotope balance equations show quantitatively how the isotopically labeled concentration of a metabolic pool changes over time, with the general form dP ∗ P∗ M∗ = Vin − Vout dt M P

(4.2)

where each M is the metabolic intermediate that provides mass for the pool at the rate Vin . Because 13 C has been shown to have a negligible isotope effect (Attwood et al., 1986; Melzer and Schmidt, 1987; Tipton and Cleland, 1988), the rate of isotopic inﬂow from each source is equal to the rate of mass (labeled plus unlabeled) ﬂow from that source, multiplied by the fraction of the ﬂow that is 13 C-labeled.

4.3.2. Constraints upon a Model A model such as the one shown in Figure 4.3 typically has more parameters than it would be possible to evaluate in a typical set of experiments. Even if it were possible to derive some information about all of the parameters separately in the model, the ability to place constraints on the system allows one to supply more information in the model, which decreases the uncertainty in those parameters that are ﬁtted. Therefore, it is often necessary and always desirable to constrain the values of model parameters within reasonable limits. For example, in the model of Figure 4.3, it is known that infusing C1-labeled glucose does not change the concentrations of brain glutamate or glutamine. Therefore, we can supply mass balance constraints on the ﬂows into and out of glutamate and glutamine C4 and C3. Therefore, Vgln = Vcycle + Vefﬂux , and Vefﬂux = Vana .

4.3.3. Simpliﬁcations of a Model The model of Figure 4.3 has been simpliﬁed in a variety of ways, and the effect of such simpliﬁcations can be calculated using the metabolic simulations. One of the simpliﬁcations was the grouping of metabolic intermediates that are in rapid equilibrium, such as glucose-6-phosphate and fructose-6-phosphate. To evaluate the effect of grouping intermediates, the time courses were simulated for several values of Vtca , assuming no exchange between the intermediate metabolites. Simulated labeling time courses were then used as input for the model’s ﬁtting procedure. The values of Vtca derived from the simulated time courses assuming no exchange among intermediates were overestimated, but the overestimate was less than 3 % of the simulation value of Vtca . Similar procedures have been applied to evaluate the assumption of lactate–pyruvate exchange being very fast, the effects of uncertainty in glucose transport kinetics, blood–brain exchange of pyruvate and lactate, and other simpliﬁcations of metabolic models.

4.3.4. Isotopomer Analysis in a Model 13

C can be incorporated not only at different positions of a molecule, but simultaneously in different combinations of positions in the molecules. The term isotopomers represents a single chemical species that is labeled with the same number of isotopes but at different locations within the molecule. In the analysis of metabolic NMR data, isotopomers is more loosely used to represent differently labeled moieties of the same chemical, no matter how many locations are labeled in the molecule. The evaluation of isotopomers has been used powerfully to quantify proportions of ﬂows at junctions of different metabolic ﬂows (den Hollander et al., 1979; Sherry et al., 1988). Generally, the measurements are made at an isotopic steady state and analyzed using the proportion of one isotopomer to another. One example is the application of isotopomers to quantify the proportions of labeled and unlabeled carbon ﬂowing to glutamate during an infusion of [1-13 C]glucose through the metabolic schematic shown

62

Brain Energetics and Neuronal Activity

in Figure 4.3. Because citrate and oxaloacetate are so much smaller than the glutamate and glutamine, their kinetic effects can be neglected. Therefore, the isotope balance equations are dGlu4 dt dG ln4 dt dGlu3 dt dG ln3 dt dGlu3,4 dt dG ln3,4 dt

= = = = = =

G ln4 Glu4 L3 Vgly + Vcycle + (0)Vdil − (Vtca + Vg ln ) L G ln G ln Glu4 G ln4 Vg ln − (Vcycle + Vefﬂux ) Glu G ln 1 1 Glu4 1 Glu3 G ln3 Glu3 1 L3 Vana + (0)Vana + Vtca,net + Vtca,net + Vcycle − (Vtca + Vg ln ) 2 L 2 2 Glu 2 Glu G ln Glu G ln3 Glu3 Vg ln − (Vcycle + Vefﬂux ) Glu G ln L3 Glu3 G ln3,4 Glu3,4 Vtca + Vcycle − (Vtca + Vg ln ) L Glu G ln Glu Glu3,4 G ln3,4 Vg ln − (Vcycle + Vefﬂux ) Glu G ln

When the system is studied at a metabolic steady state, in which condition the concentrations of metabolites are not changing, Vgln = Vcycle + Vefﬂux , and Vefﬂux = Vana . So, at an isotopic steady state, Glu∗4 G ln∗4 = , G ln Glu and

G ln∗3,4 Glu∗3 G ln∗3 Glu∗3,4 = , = Glu Glu Glu Glu

Glu4 L3 Vtca − Vdil = Glu L Vtca

The equation for [3,4-13 C2 ]glutamate, with substitution of Glu4 /Glu, yields Vdil Glu3 Glu4 =1− Vtca Glu34 Therefore, from measurements of the concentration of C3-labeled glutamate, C4-labeled glutamate, and glutamate labeled at both C3 and C4, one can determine the proportion of the TCA cycle that is fueled by an unlabeled carbon source.

4.4. MECHANICS OF FITTING DATA Fitting a model to experimental data is a procedure that answers two questions. (1) Can the values of parameters in this model be adjusted to provide an adequate simulation of the observations? (2) If this model were true, what values of the parameters are needed to explain the observations? In practice, the parameters are adjusted until some tolerance is attained for the proximity of the data and the simulation.

4.4.1. Least-squares Fitting The most common measurement of proximity of the data and simulation is least-squares ﬁtting. Leastsquares ﬁtting relies on the sum of the square of the difference between the data and the simulation. For a function f (t) that uses m parameters a1 , a2 , a3 , . . . am , χ2 =

n i=1

[f (a1 ..m , ti ) − yi (ti )]2

(4.3)

Metabolic Modeling Analysis of Brain Metabolism

63

where yi (ti ) is the ith experimental observation, and f (a1 ..m , ti ) is the simulated function calculated at t = ti . The least-squares ﬁtting is used to ﬁnd the set of values of a1 ..m that yields the minimum value of χ 2 . For a straight line, least-squares ﬁtting yields the values of two parameters, which are the slope a1 and intercept a2 . The purpose of the regression analysis is to ﬁnd the best ﬁt of the function yi = a1 ti + a2 . The ﬁtting is performed based on the assumption that the minimum value of χ 2 is obtained when

and

n ∂χ 2 ∂f (a1 , a2 , ti ) =2 [f (a1 , a2 , ti ) − yi (ti )] = 0 ∂a1 ∂a1 i=1

(4.4)

n ∂f (a1 , a2 , ti ) ∂χ 2 =2 [f (a1 , a2 , ti ) − yi (ti )] = 0 ∂a2 ∂a2 i=1

(4.5)

For a straight line, there are analytic expressions for the values of a1 and a2 , which are y a1 =

n

ti2 − t

i=1 n

n

ti yi

i=1

ti2

− nt

(4.6)

2

i=1

and n(t)(y) − a2 = 2

nt −

n

i=1 n

ti yi (4.7)

ti2

i=1

Therefore, a1 and a2 are explicitly determined and result in a straightforward solution. For most problems of metabolic modeling, however, there are no explicit expressions for the best values of the ﬁtted parameters, so they must be evaluated iteratively. A variety of methods exist for the iterative determination of model parameters.

4.4.2. Numerical Fitting Algorithms Algorithms exist that search for the parameter set that provides the best approximation to the data, and the performance of each varies with respect to the time to complete the estimation and the likelihood of success. In general, the sum of squares of the differences, called χ 2 , can be imagined as a surface whose dimensions are the ranges of the values of the parameters in the model. The coordinates of each point on the surface are a set of values of the model parameters, and the height of the surface is the value of χ 2 that occurs for those parameter values (Figure 4.5). Because some combinations of points give a better approximation to the data than other combinations, the surface will have bumps and dips, with one dip that is deeper than any of the others. The coordinates of that deepest dip are the least-squares values of the model for the data. Fitting algorithms are assigned the task of locating that deepest dip. Examining Figure 4.5, one sees many shallow dips, which are called false minima, and if a search focuses quickly on one of these minima without checking for others, the conclusions will be incorrect. One can also see that if a search begins too far away from the true minimum, with a small search range, the algorithm will not ﬁnd the true minimum. Search algorithms are designed to balance speed and robustness in the search.

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c2

B

A

Figure 4.5. χ 2 surface for a model f (A, B), which is the sum of the squares of the difference between the model prediction and the actual data for all combinations of the ﬁtted parameters A and B. The value of χ 2 is a function of the ﬁtted parameters in a model, which are in this case a1 and a2 . A least-squares ﬁtting algorithm ﬁnds the combination of ﬁtted parameters that yields the minimum value of χ 2 . The graph shows one deep minimum and two shallower minima that are called false minima. Most problems require an iterative search for the true minimum value of χ 2 . Greedy search algorithms may ﬁnd only a false minimum, while a more robust method that is more likely to ﬁnd the true minimum may take longer to complete the search.

An algorithm is called greedy if it settles quickly for the ﬁrst minimum that it encounters in the χ 2 surface, but an algorithm that searches too many possibilities would be impractically slow. The simplex algorithm tends to be quick but greedy, by comparing the amplitudes of χ 2 at several different coordinates of the χ 2 surface. Simulated annealing, on the other hand, rarely fails but takes much longer to run. Simulated annealing can be implemented in many ways, but the common feature is the incorporation of a random alteration of the test coordinates of the χ 2 surface. In simulated annealing, the random alteration is large at the beginning, analogous to the vibrations of atoms in a crystal lattice, and as the ﬁt progresses, the random changes become smaller and smaller until the ﬁt is nearly identical to one of the conventional ﬁtting methods such as simplex. A method that is faster than simulated annealing and still effective is the Levenberg–Marquardt algorithm, which uses not only the amplitude but also the slope of χ 2 at various locations. The Levenberg–Marquardt algorithm is the most common ﬁtting algorithm in use today for the analysis of kinetic 13 C NMR data, but when it does fail, simulated annealing has been able to ﬁll the need.

4.4.3. Effects of Scatter When model parameters are estimated from 13 C NMR data, there arises the question of how well the values are known, given the uncertainty in the experimental data. For the case of studies that are done in vivo, one wants to know not only the group mean and standard deviation, but whether the individual variability is greater than is expected given the group statistics. That is, if a study shows a twofold difference in the rate of a pathway across individuals, can that be explained on the basis of measurement error, or is there a strong inter-subject variability? On the other hand, for time courses made from tissue samples, with individual time points obtained in separate animals or groups of animals, then the group statistics must

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be derived from the scatter in the time course. Such an assumption convolves the noise from the NMR measurements and differences in the tissue samples. Most software provides an estimate in the uncertainties of parameters based on a calculation that uses the fundamental theorem of calculus, which includes an assumption of vanishingly small noise. For many NMR spectroscopic experiments, the noise is not vanishingly small and is frequently 10–30 % of the steady-state isotopic labeling, depending on how the study is done and what metabolites are measured. Having violated the assumption that underlies the manner in which most analyses determine uncertainties in ﬁtted parameters, we are left to ﬁnd an alternative and to answer the question of how small noise must be if it is to be called ‘vanishingly small’. One solution to the problem of non-negligible noise has been the implementation of the Monte Carlo estimation of scatter. In the Monte Carlo approach, noise with the same Gaussian distribution is added randomly to the least-squares ﬁt to create a simulated, noisy data set, and that simulated set is fed into the model to derive a simulated set of parameters (Mason et al., 1995; Mason et al., 1992b). The process of generating simulated sets is repeated a hundred or a thousand times to generate a list of simulated ﬁtted parameters that are used to create a histogram of the distribution of the ﬁtted parameters based on the scatter in the original data. The list of simulated parameters can be used to derive a standard deviation, and the histogram can be used to evaluate whether or not the parameter’s value has a normal distribution of uncertainty. As to the size of noise that violates the assumption, it depends on the model that is evaluated. The reliability of the calculus-based determinations can be evaluated by comparison of the standard methods, which include the assumption, and the Monte Carlo approach, which does not. As shown in Figure 4.6, 1200 Linear regression

1000 Freq

800

5%

600 400

10% 20%

200 0 0.09

0.095

0.1 Slope

0.105

0.11

Single exponential

1200 5% Freq

900 600 10%

300

20% 0

0

0.1

0.2

0.3

0.4

Rate (µmol/min/g)

Figure 4.6. Relationships between the level of noise and the distribution of uncertainty for (top) the slope in linear regression analysis and (bottom) the rate constant obtained with least-squares ﬁtting of a single-exponential function (bottom). Linear regression analysis maintains its ideal, normal distribution of uncertainty even with high levels of noise, whereas a single-exponential function rapidly deviates from the common theoretical treatment of a normal distribution of uncertainty.

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Freq

400

Monte-Carlo

300 200

Standard normal

100 0

0

0.1

0.2

0.3

0.4

Rate (µmol/min/g)

Figure 4.7. Comparison of distributions of normal and general Monte Carlo estimations of uncertainty for the rate determined from a single-exponential function for noise = 20 % of the steady-state value of the exponential.

the traditional error estimations from the linear regression analysis of Equations (4.1) and (4.2) track the Monte Carlo estimations indistinguishably across a wide range of noise levels. In contrast, the standard calculus-based error estimation for the rate in a single-exponential equation, which is similar to the form of the 13 C-labeling time course of glutamate C4, deviates slightly from the ideal, normal distribution even when the noise exceeds 5 % of the steady-state signal amplitude. For a steady-state signal-to-noise level of 20 %, the deviation becomes severe. When distributions are skewed as shown in Figure 4.6, the common estimates of noise express the uncertainty inadequately, as a standard deviation, which does not indicate the skewedness of the distribution, and that standard deviation is an underestimate of the true width of the distribution. The effects of misapplying the standard distribution estimates can yield either false positives or false negatives, depending on how the data are distributed. Figure 4.7 shows the true distribution for the rate derived using a single-exponential function together with the idealized estimate of the normal distribution. If the derived rate is compared to zero, the normal distribution leads to a false conclusion that the rate does not differ signiﬁcantly from zero. In contrast, if the derived rate is compared to a value of 0.25 µmol/min/g, the normal distribution leads to a false conclusion that the derived rate does differ signiﬁcantly. The practical reason for the skewedness is important to consider in the context of 13 C-labeling studies. Suppose that one looks at the value of y = 1 − e−kt where t = 20 min. For k = 0, 0.03, 0.06, 0.09, and 0.12, the value of y is 0, 0.45, 0.70, 0.83, and 0.91, which means that as the value of k rises, a given increment of noise in the data requires larger and larger values of k to achieve the same ﬁt. So it goes in general with metabolic rate measurements, where the difference in the effects of two fast rates is not generally as great as the difference between two slower rates.

4.4.4. Information Content in a

13

C-Labeling Experiment

Kinetic 13 C MR studies are performed by observing the appearance of the 13 C isotope over time in the sample of interest, and the entire time course is analyzed to obtain all of the information that is desired from the experiment. However, not all of the data points contain the same quantity of information about each parameter, and if an investigator knows which points are the most important in a study, it is sometimes possible to improve the experiment design. Therefore, it can be useful to understand how the information is distributed and how much information in total is obtained within the set of data. To

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0.03 0.02

dk/dy

Sensitivity

0.01 0.00 −0.01 −0.02

dE/dy

−0.03

Percent enrichment

−0.04 1.0 0.8 0.6 0.4 0.2 0.0

0

20

40 Time (min)

60

Figure 4.8. Sensitivities and enrichment for a single-exponential model of 13 C-labeling. Top: sensitivities of the ﬁtted values of the metabolic rate constant k and the source enrichment E as functions of time for a 60 min measurement of the time course of 13 C-labeling of the product P. Bottom: time course of the percent enrichment corresponding to a single-exponential model.

arrive at a quantitative understanding of information, it is necessary to introduce the terms sensitivity, data deﬂection, and relevance. Sensitivity is the inﬂuence that a data point exerts on the estimated value of a ﬁtted parameter. That is, the sensitivity of a particular data point is the amount of change in the ﬁtted parameter for a given change in the amplitude of the data point. Speciﬁcally, we can examine this idea with the single-exponential expression that was used for the previous example, where the respective sensitivities of the ith data point to the rate constant k = V /P and to the source enrichment E are dk/dyi and dE/dyi . We can estimate k/yi and E/yi for i = 1 to n data points using values of yi that are small enough to approximate dk/dy and dE/dy, in this case y = 0.01E (Figure 4.8). The noise sensitivity is how much the parameters change with respect to the level of noise, or knoise /yi,noise and Enoise /yi,noise . The data deﬂection of a data point is how much the point is moved over the ranges of possible values of the ﬁtted parameters, which in the present example are V and E. They are approximated as yi,deﬂection /Edeﬂection and yi,deﬂection /kdeﬂection . The relevance is deﬁned as the reciprocal of the product of the noise sensitivity and the true deﬂection, minus one, or yi,deﬂection yi,deﬂection knoise Enoise RV ,i = × − 1 and RE,i = × −1 (4.8) kdeﬂection yi,noise Edeﬂection yi,noise A relevance ratio of 0 or less for V/P means that despite any sensitivity of the value of the parameter V to the amplitude of the data, the noise obscures the relevant effects and there is no information about V at that the ith data point. Besides a point-by-point analysis, the relevance ratio can be calculated for the

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entire data set, as well, according to n n yi,deﬂection 2 knoise 2 RV ,i = × − 1, kdeﬂection yi,noise i=1 i=1 RE,i

n n yi,deﬂection Enoise 2 = −1 × Edeﬂection yi,noise i=1 i=1

(4.9)

The grouped approach in Equation (4.9) allows the amount of information in studies to be compared quantitatively, taking into account the sensitivity of the measurement and the level of noise in the study. The individual approach that is embodied by Equation (4.8) allows the amount of information to be compared not only across studies but among the time points in an individual experiment. Consider, for example, the two-compartment model of Figure 4.1. Two methods to measure the rate of anaplerosis relative to the rate of glutamine synthesis are (1) the measurement of the time courses of isotopic labeling of glutamate and glutamine C4 during an infusion of [1-13 C]glucose (Gruetter et al. 2001; Shen et al., 1999) and (2) the measurement of steady-state glutamate and glutamine C2 and C3 labeling while supplying [2-13 C]glucose (Kanamatsu and Tsukada 1999; Sibson et al., 1998; Taylor et al., 1996). Figures 4.9 and 4.10 show the sensitivities and time courses for the ratio of Vana /Vgln over time for the [1-13 C]glucose and [2-13 C]glucose measurements. It is clear that most of the information sensitivity about From [1-13C]Glucose

Percent enrichment

d (Vana/Vgln)/dy

0.3

Steady state

0.2 0.1

Glu C4

0.0 −0.1 35 30 25 20 15 10 5 0

Gln C4

Glu C4 (0.09) Glu C4 (0.18) Gln C4 (0.09) Gln C4 (0.18) 0

25

50

75

100

Time (min)

Figure 4.9. Time courses of 13 C-labeling of glutamate and glutamine and the sensitivities of the ratio of Vana /Vgln to the observed 13 C enrichments of glutamate and glutamine during infusions of [1-13 C]glucose. The simulations were done for Vana /Vgln = 0.09 and 0.18, and the shaded portion covers the time when the labeling approaches an isotopic steady state. Bottom: the lack of difference in labeling with the two values of Vana /Vgln demonstrate a low sensitivity of the data to Vana /Vgln . Top: for times less than 15 min after the start of the glucose infusion, positive deviations in the glutamine C4 enrichment lead to higher values for Vana /Vgln , and after that, the opposite is true. The steady-state portion of the measurement shows almost no difference in the time courses, and that fact is reﬂected in the low steady-state sensitivity shown in the top graph.

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From [2-13C]Glucose

Percent enrichment

d (Vana/Vgln)/dy

0.2

Steady state

Gln C2

0.1 0.0 −0.1

Glu C2

−0.2

Gln C2 (0.18)

4

.09)

3

Gln C2 (0

.18) Glu C2 (0 Glu C2 (0.09)

2 1 0

0

25

50

75

100

Time (min)

Figure 4.10. Time courses of 13 C-labeling of glutamate and glutamine and the sensitivities of the ratio of Vana /Vgln to the observed 13 C enrichments of glutamate and glutamine during infusions of [2-13 C]glucose. The simulations were done for Vana /Vgln = 0.09 and 0.18, and the shaded portion covers the time when the labeling approaches an isotopic steady state. Bottom: there is a marked difference in labeling with the two values of Vana /Vgln , demonstrating a low sensitivity of the data to Vana /Vgln . Accordingly, the sensitivity to Vana /Vgln is high (top). The steady-state portion of the measurement shows a large difference, primarily in the C2-labeling of glutamate.

Vana /Vgln resides in the early parts of the C1-glucose study, although the sensitivity to noise is so high at that point that there is little relevance of the measurement to those parameters. In contrast, the C2-glucose study shows a distribution of sensitivity and relevance across the time course, with a large quantity of information available during the isotopic steady-state portion of the measurement. The ability to acquire the data at isotopic steady state means that a subject can be infused in relative comfort, outside the scanner, entering the magnet once an isotopic steady-state is achieved, the measurement can go on for an hour or even more, depending on how long a subject can be kept comfortably in the scanner. On an individual basis for human studies, the scatter in glutamate and glutamine C4 during are typically 1.7 and 3.8 % in 5 min of accumulation of signal, and the relevances of Vana /Vgln for the time-dependent [1-13 C]glucose study and [2-13 C]glucose steady-state measurements are −0.68 and +0.85, respectively. On a subject-by-subject basis, it should be possible to measure the value of Vana /Vgln using the [2-13 C]glucose protocol. In contrast, the C1-labeled glucose protocol has no relevance for individual subjects. If the noise level were reduced by four- to ﬁve-fold, the [1-13 C]glucose protocol would have some relevance for measuring Vana /Vgln .

4.5. CONCLUSION In this review we have covered the basic concepts and issues in ﬁtting 13 C NMR data obtained from the brain in vivo to determine metabolic rates. The limiting factors in determining metabolic rates with 13 C MRS are sensitivity and the deconvolution of multiple metabolic compartments. As shown above, it is useful to evaluate distributions of uncertainty in the ﬁtted parameters. This feature is important because of effects of non-normal distributions of uncertainty when analyzing data. When working in vivo, it is common to obtain skewed distributions of uncertainty, which can cause a group’s standard deviation to be

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overestimated and obscure signiﬁcant differences. By estimating the effects of noise on the distributions of uncertainty before performing most of the experiments in a set of studies, one might identify speciﬁc portions of the experimental protocol that should be improved to narrow the distribution of uncertainty to the necessary precision. Mathematical modeling provides ways to quantify which are the most sensitive portions of a 13 C isotopic labeling study. A ﬁtted variable usually has unequal sensitivities to the 13 C-labeling time courses of different metabolites throughout a kinetic labeling study, and a study may be designed to emphasize the data acquisition in the most sensitive portions of an infusion protocol, while de-emphasizing the less sensitive portions of the study. A mathematical evaluation of the sensitivity also permits the calculation of the relevance of an experiment, where the relevance is a measurement of the ability of a study to provide a meaningful estimate of the value of a parameter from a ﬁt to the isotopic time course data. The relevance provides an objective way to compare the effectiveness of various experimental protocols.

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K¨unnecke, B., Cerd´an, S., and Seelig, J. (1993) Cerebral metabolism of [1,2-13 C2 ]glucose and [U-13 C4 ]3-hydroxybutyrate in rat brain as detected by 13 C NMR spectroscopy. NMR Biomed 6: 264–277 Lebon, V., Petersen, K. F., Cline, G. W., Shen, J., Mason, G. F., Dufour, S., Behar, K. L., Shulman, G. I., and Rothman, D. L. (2002) Astroglial contribution to brain energy metabolism in humans revealed by 13C nuclear magnetic resonance spectroscopy: elucidation of the dominant pathway for neurotransmitter glutamate repletion and measurement of astrocytic oxidative metabolism. J Neurosci 22: 1523–1531 Lewandowski, E. D., Yu, X., LaNoue, K. F., White, L. T., Doumen, C., and O’Donnell, J. M. (1997) Altered metabolite exchange between subcellular compartments in intact postischemic rabbit hearts. Circ Res 81: 165–175 Mason, G. F., Behar, K. L., Rothman, D. L., and Shulman, R. G. (1992a) NMR determination of intracerebral glucose concentration and transport kinetics in rat brain in vivo. J Cereb Blood Flow Metab 12: 448–455 Mason, G. F., Falk Petersen, K., de Graaf, R. A., Kanamatsu, T., Otsuki, T., and Rothman, D. L. (2003) A comparison of 13 C NMR measurements of the rates of glutamine synthesis and the tricarboxylic acid cycle during oral and intravenous administration of [1-13 C]glucose. Brain Res Protocols. 10(3): 181–90 Mason, G. F., Gruetter, R., Rothman, D. L., Behar, K. L., Shulman, R. G., and Novotny, E. J. (1995) Simultaneous determination of the rates of the TCA cycle, glucose utilization, alpha-ketoglutarate/glutamate exchange, and glutamine synthesis in human brain by NMR. J Cereb Blood Flow Metab 15: 12–25 Mason, G. F., Martin, D. L., Martin, S. B., Manor, D., Sibson, N. R., Patel, A., Rothman, D. L., and Behar, K. L. (2001) Decrease in GABA synthesis rate in rat cortex following vigabatrin administration correlates with the decrease in GAD67 protein. Brain Res 914: 81–91 Mason, G. F., Rothman, D. L., Behar, K. L., and Shulman, R. G. (1992b) NMR determination of TCA cycle rate and α-ketoglutarate/glutamate exchange rate in rat brain. J Cereb Blood Flow Metab 12: 434–447 Melzer, E., and Schmidt, H.-L. (1987). J Biol Chem 262: 8159 Moreno, A., Bluml, S., Hwang, J. H., and Ross, B. D. (2001a) Alternative 1-(13)C glucose infusion protocols for clinical (13)C MRS examinations of the brain. Magn Reson Med 46: 39–48 Moreno, A., Ross, B. D., and Bluml, S. (2001b) Direct determination of the N-acetyl-L-aspartate synthesis rate in the human brain by (13)C MRS and [1-(13)C]glucose infusion. J Neurochem 77: 347–350 Pardridge, W. M., and Oldendorf, W. H. (1977) Transport of metabolic substrates through the blood-brain barrier. J Neurochem 28: 5–12 Rothman, D. L., Behar, K. L., Hyder, F., and Shulman, R. G. (2003) In vivo NMR studies of the glutamate neurotransmitter ﬂux and neuroenergetics: implications for brain function. Annu Rev Physiol 65: 401–427 Rothman, D. L., Novotny, E. J., Shulman, G. I., Howseman, A. M., Petroff, O. A. C., Mason, G., Nixon, T., Hanstock, C. C., Prichard, J. W., and Shulman, R. G. (1992) 1 H-[13 C] NMR measurements of [4-13 C]glutamate turnover in human brain. Proc Natl Acad Sci USA 89: 9603–9606 Shen, J., Petersen, K. F., Behar, K. L., Brown, P., Nixon, T. W., Mason, G. F., Petroff, O. A., Shulman, G. I., Shulman, R. G., and Rothman, D. L. (1999) Determination of the rate of the glutamate/glutamine cycle in the human brain by in vivo 13 C NMR. Proc Natl Acad Sci USA 96: 8235–8240 Shephard, G. M. (1994) The Synaptic Organization of the Brain. Oxford: Oxford University Press Sherry, A. D., Malloy, C. R., Roby, R. E., Rajagopal, A., and Jeffrey, F. M. (1988) Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy. Biochem J 254: 593–598 Sibson, N. R., Dhankhar, A., Mason, G. F., Rothman, D. L., Behar, K. L., and Shulman, R. G. (1998) Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proc Natl Acad Sci USA 95: 316–321 Sibson, N. R., Mason, G. F., Shen, J., Cline, G. W., Herskovits, A. Z., Wall, J. E. M., Behar, K. L., Rothman, D. L., and Shulman, R. G. (2001) In vivo 13C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle ﬂux in rat brain during [2-13C]glucose infusion. J Neurochem 76: 975–989 Sillerud, L. O., Alger, J. R., and Shulman, R. G. (1981) High-resolution protein NMR studies of intracellular metabolites in yeast using 13 C decoupling. J Magn Reson, Ser B 45: 142–150 Sonnewald, U., Gribbestad, I. S., Westergaard, N., Krane, J., Unsg˚ard, G., Petersen, S. B., and Schousboe, A. (1991) First direct demonstration of preferential release of citrate from astrocytes using [13 C] NMR spectroscopy of cultured neurons and astrocytes. Neurosci Lett 128: 235–239 Sonnewald, U., Westergaard, N., Jones, P., Taylor, A., Bachelard, H. S., and Schousboe, A. (1996) Metabolism of [U13C5] glutamine in cultured astrocytes studied by NMR spectroscopy: ﬁrst evidence of astrocytic pyruvate recycling. J Neurochem 67: 2566–2572

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Szczepaniak, L., Babcock, E. E., Malloy, C. R., and Sherry, A. D. (1996) Oxidation of acetate in rabbit skeletal muscle: detection by 13C NMR spectroscopy in vivo. Magn Reson Med 36: 451–457 Taylor, A., McLean, M., Morris, P., and Bachelard, H. S. (1996) Approaches to studies on neuronal/glial relationships by 13C-MRS analysis. Dev Neurosci 18: 434–442 Tipton, P. A., and Cleland, W. W. (1988). Biochemistry 27: 4325 Van den Berg, C. J., and Garﬁnkel, C. D. (1971) A simulation study of brain compartments: metabolism of glutamate and related substances in mouse brain. Biochem J 123: 211–218 Van den Berg, C. J., Krzalic, L. J., Mela, P., and H. Waelsch. (1970) Compartmentation of glutamate metabolism in brain: Evidence for the existence of two different tricarboxylic acid cycles in brain. Biochem J 113: 281–290 Waagepetersen, H. S., Sonnewald, U., Larsson, O. M., and Schousboe, A. (2000) Compartmentation of TCA cycle metabolism in cultured neocortical neurons revealed by 13C MR spectroscopy. Neurochem Int 36: 349–358 Yu, X., White, L. T., Doumen, C., Damico, L. A., LaNoue, K. F., Alpert, N. M., and Lewandowski, E. D. (1995) Kinetic analysis of dynamic 13C NMR spectra: metabolic ﬂux, regulation, and compartmentation in hearts. Biophys J 69: 2090–2102

Section B Neuroenergetics and Activity

5 Cerebral Energetics and Neurotransmitter Fluxes Nicola R. Sibson Experimental Neuroimaging Group Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

5.1

13

C MRS Measurements of Cerebral Energy Metabolism 5.1.1 Neuronal Energy Metabolism 5.1.2 Contributions of Astrocytes and GABAergic Neurons to Cerebral Energy Metabolism 5.2 MRS Measurements of Neurotransmitter Fluxes 5.2.1 Measuring the Glutamate–Glutamine Cycle 5.2.2 Validating the 13 C MRS Measurement of Glutamate–Glutamine Cycling 5.3 Energetic Requirements of Neurotransmission 5.3.1 Glutamate as a Mediator of Neurometabolic Coupling 5.3.2 Implications for Studies of Human Brain 5.4 Summary

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One of the major advantages of MRS is that reaction kinetics, and hence metabolic rates, can be measured noninvasively in vivo. This ability of MRS particularly lends it to biochemical studies of the brain which are largely inaccessible in vivo by other methodologies. 13 C MRS, in particular, has been used extensively in recent years to measure the rate of cerebral oxidative glucose consumption in vivo and, more recently, to measure certain neurotransmitter ﬂuxes (glutamate, GABA) that are intrinsically linked to neuronal activity. The term ‘neuronal activity’ applies to a spectrum of energy requiring processes, which include not only neurotransmitter release, uptake and recycling, but also action potential propagation, restoration Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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and maintenance of membrane potentials, and vesicular recycling. Consequently, it is to be expected perhaps that neuronal activity and energy metabolism should be tightly coupled in the brain, and indeed it was over a century ago that Sherrington ﬁrst suggested that such a coupling exists. Nonetheless, the energetic cost of neuronal activity, and the fraction of total cerebral energy consumption that this represents, has long been debated and is only now being resolved. Furthermore, the relationship between neuronal activity and energy metabolism has implications for the interpretation of data from functional neuro-imaging studies, which mostly rely on an implied relationship between the measured parameters (glucose consumption, oxygen consumption or blood ﬂow) and neuronal activity. The 13 C MRS studies discussed in this chapter have begun to provide answers to some of these key biochemical questions that have remained elusive for many decades. This chapter covers recent in vivo 13 C MRS studies in which measurements of cerebral energy metabolism and neurotransmitter ﬂuxes have been made, and which address the issue of the relationship between neuronal activity and cerebral energetics. The majority of studies that are discussed have been performed in rat brain, but where analogous studies in human brain are available these are also considered.

5.1.

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C MRS MEASUREMENTS OF CEREBRAL ENERGY METABOLISM

As discussed in the introductory chapter, glucose is the primary substrate for energy metabolism in the brain in vivo (1). Each glucose molecule undergoes nonoxidative (anaerobic) glycolysis to yield two molecules of pyruvate, which are subsequently oxidized to CO2 via the tricarboxylic acid (TCA) or Krebs cycle. The oxidative steps of glucose metabolism are generally considered to contribute ∼36 of a total of ∼38 high-energy ATP molecules generated during metabolism of a single glucose molecule, whilst just two ATP molecules are contributed by the glycolytic pathway. Approximately 15 % of glucose that undergoes glycolysis is converted to lactate and does not enter the TCA cycle, although this may be balanced by a corresponding uptake and oxidative metabolism of ketone bodies. Techniques by which cerebral glucose metabolism (CMRglc ) can be measured, either globally or regionally, have been available for some time – either by snapshot in vivo techniques such as autoradiography or true in vivo techniques such as arterio-venous difference measurements or positron emission tomography (PET). However, it is highly likely that the energetic requirements of the various cell populations of the brain will differ considerably in their energetic requirements and, hence, metabolic rates. Consequently, there is a need for measurement methods that can speciﬁcally target the different cell populations in the brain. It is at this level that in vivo 13 C MRS comes into its own, primarily as a consequence of two main factors – ﬁrstly, the chemical speciﬁcity of the technique by which the chemical shift of each 13 C peak identiﬁes a speciﬁc carbon position within a particular molecule, and, secondly, the restricted localization of certain enzymes to speciﬁc cell types. Glutamate is the major excitatory neurotransmitter in the mammalian brain, whilst γ -aminobutyric acid (GABA) is the primary inhibitory transmitter. In contrast, glutamine is synaptically inert, yet plays a key role in the metabolism of both neurotransmitters. The brain pools of glutamate, GABA and glutamine have been shown to be localized within glutamatergic neurons, GABAergic neurons, and astrocytes, respectively (under nonpathological conditions), and it is the localization to speciﬁc cell types of key enzymes involved in the metabolism of these molecules that enables 13 C MRS to separate the energetic requirements of these cell populations. In this section, we will discuss how 13 C MRS can be used to measure pathways of energy metabolism in the cerebral cortex.

5.1.1. Neuronal Energy Metabolism As explained in previous chapters, the general approach adopted to measure metabolic rates by 13 C MRS is to intravenously infuse a 13 C-labeled substrate (e.g. glucose, acetate) which will easily pass through the blood–brain barrier and be metabolized within the brain. Over time, a variety of metabolite pools within the brain become enriched with 13 C, and these can be detected by 13 C MRS. A number of studies of human

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Figure 5.1. Example of a 1 H-decoupled, NOE-enhanced 13 C MRS spectrum acquired from human cerebral cortex over a period of ∼60 min during infusion of [1-13 C]glucose. Resonances from several key metabolites coupled to energy and neurotransmitter metabolism are visible including, glutamate, glutamine, GABA, aspartate and NAA.

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brain (2–5) have now demonstrated that several key metabolites coupled to brain energy metabolism can be detected using 13 C MRS, including glutamate, glutamine, GABA, aspartate and NAA (see Figure 5.1). As described in Chapter 4, the development of metabolic models has enabled determination of absolute metabolic rates from the time courses of isotopic labeling in the brain. The most commonly used isotopic precursor for both 13 C and 1 H– 13 C MRS studies is [1-13 C]glucose. The original MRS measurements of metabolic ﬂuxes in the brain in vivo were of the rates of the tricarboxylic acid (TCA) cycle and glucose oxidation using 1 H– 13 C MRS to follow the ﬂux of 13 C isotope from [113 C]glucose into cerebral metabolites (6). The [1-13 C]glucose is initially metabolized to pyruvate via the glycolytic pathway, which results in labeling of pyruvate at C-3. The label is then transferred to the TCA cycle by the sequential actions of pyruvate dehydrogenase (PDH) and citrate synthase. When the label reaches α-ketoglutarate (C-4) it is transferred to glutamate (C-4) by the high activity exchange reactions of the amino acid transaminases and mitochondrial/cytosolic transporters. Since the exchange activity of these transaminases is rapid, labeling of the α-ketoglutarate and glutamate pools is in close equilibrium. Thus, the ﬂux of 13 C isotope from [1-13 C]glucose into [4-13 C]glutamate (shown schematically in Figure 5.2(A)) enables quantitative determination of the TCA cycle rate (2, 3, 5, 7–10). Since the TCA cycle is intrinsically linked to oxidative phosphorylation, and glucose is the primary fuel of oxidative metabolism in the brain, the measurements of the TCA cycle may be converted to measurements of glucose oxidation using known stoichiometries (7, 9). However, we now come to the question of which speciﬁc cell type, or group of cell types, are we measuring the metabolism of? All of the different cell populations of the brain contain the necessary enzymes for glycolysis, the TCA cycle and oxidative phosphorylation. Consequently, one might assume that the rate of oxidative glucose metabolism that is obtained by this measurement is an average of the metabolic rates of all cell types. However, since the measurement is made by following the ﬂux of 13 C label into glutamate the weighting of this measurement to the various cell types is determined to a large extent by the relative pool sizes of glutamate in those different cell populations. The existence of more than one glutamate pool in the brain was determined several decades ago in 14 C tracer studies (11), and initially two primary glutamate pools were identiﬁed, one large and one small. Based on kinetic and immunohistochemical staining studies the large glutamate pool is believed to correspond to the glutamate pool in glutamatergic neurons (7, 12, 13), whilst the small one has been shown to be associated with glial-speciﬁc enzymes and pathways. The difference in sizes of these pools is of the order of 100-fold, and, therefore, the glutamate pool observed by 13 C MRS is generally attributed to the large glutamate pool within glutamatergic neurons. Consequently, the weighting of the 13 C measurement of oxidative glucose consumption obtained by infusion of [1-13 C]glucose is strongly weighted towards the glutamatergic neuronal population. The rate of glucose oxidation in glutamatergic neurons has been determined in numerous studies from both 13 C and 1 H– 13 C MRS measurements of cortical glutamate labeling during infusion of [1-13 C]glucose in animals (6, 9, 14–21) and humans (2–5, 7–9, 22). Comparison of the rates of neuronal glucose oxidation (0.25–0.50 µmol/min/g) measured in these studies with conventional arterio-venous difference and PET measurements of total glucose consumption indicates that the major fraction (60–90 %) of total glucose oxidation is associated with glutamatergic neurons in both the rat and human brain.

5.1.2. Contributions of Astrocytes and GABAergic Neurons to Cerebral Energy Metabolism However, as mentioned above, glutamate is present in all brain cells including astrocytes, and also GABAergic neurons. Although the concentrations of glutamate in these other cell types are considerably lower than in glutamatergic neurons, the contributions of these cell populations to cerebral glucose metabolism cannot

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be entirely ignored. The rate of glucose oxidation in astrocytes has long been a subject of controversy in studies of cerebral metabolism. Early estimates range from 10 % to over 50 % of total glucose oxidation (1). 13 C MRS may be used to measure the rate of astrocytic glucose oxidation based on the localization of the enzyme glutamine synthetase to astrocytes (23). As a result, the rate of the astrocytic TCA cycle can be calculated based on the labeling of glutamine from astrocytic glutamate. However, it is difﬁcult to obtain this metabolic rate with the commonly used [1-13 C]glucose precursor since the label enters glutamate in both neurons and astrocytes. Consequently, owing to the differences in pool sizes (neuronal glutamate ≈ 10 mM, astrocytic glutamate <1 mM), as discussed above, the labeling observed in vivo is dominated by the neuronal compartment. As a consequence, label subsequently entering glutamine is largely derived from the neuronal glutamate pool and predominantly reﬂects pathways other than the astrocytic TCA cycle (see below). A solution to this problem is to use alternatively labeled isotopic precursors that yield data speciﬁc to astrocytic metabolism. The use of alternative precursors to [1-13 C]glucose in combination with 13 C and 1 H– 13 C MRS has been common practice in both in vitro and ex vivo studies for over a decade (for review see Bachelard (24) and Cruz and Cerdan (25)) and provides a unique approach to the study of metabolic compartmentation. One isotopic precursor that has been used extensively, often in combination with [1-13 C]glucose to investigate differences between neuronal and glial metabolism, is [2-13 C]acetate. Although both neurons and astrocytes contain the enzyme for acetate catabolism, acetyl CoA synthetase, transport of acetate by astrocytes is much greater [Waniewski, 1998 #59]. Consequently, acetate is considered to be an astrocyte-speciﬁc substrate. With [2-13 C]acetate the 13 C label enters the astrocytic TCA cycle via acetyl CoA, passes into the astrocytic glutamate pool and, subsequently, into glutamine (see Figure 5.2(B)). Comparison of labeling patterns following infusion of [1-13 C]glucose and [2-13 C]acetate has yielded valuable information about cellular compartmentation and neuronal–glial interactions (26–33). Few measurements of absolute metabolic rates from such studies have been reported, although in a recent review the contribution of astrocytic glucose oxidation to total glucose oxidation was estimated to be 20–30 % (25). Consequently, the neuronal contribution was calculated to be 70–80 % of total glucose oxidation, which is similar to the ﬁndings of the in vivo studies described above. Despite the longstanding use of alternative isotopic precursors in vitro, they have only recently begun to be utilized in vivo. In a recent study of human cerebral cortex, [2-13 C]acetate was used to determine the rate of astrocytic glucose oxidation and it was found to account for ∼15 % of total glucose oxidation (34). Similar in vivo studies in rats have recently been reported using [2-13 C]glucose as the isotopic precursor (19, 35). Although [2-13 C]glucose will be metabolized by both astrocytes and neurons, this precursor acts as an astrocyte-speciﬁc precursor as a consequence of the anaplerotic pyruvate carboxylase pathway, which is localized exclusively to astrocytes in the adult brain (36–38). Via this pathway, label from [2-13 C]glucose is brought into the C-3 (and C-2) positions of the small astrocytic glutamate pool (19, 35, 36, 39), while label ﬂux through either the neuronal or astrocytic PDH pathway does not contribute to labeling in these positions (see Figure 5.2(C)). In practice, the 13 C label is ﬁrst observed in glutamine (C-3 and C-2) owing to the small size, and low MRS sensitivity, of the astrocytic glutamate and TCA cycle intermediate pools, and this labeling ﬂux can be used to obtain measurements of astrocytic metabolism. To date, absolute measurements of astrocytic metabolism have only been obtained under hyperammonemic conditions owing to the low level of isotopic labeling achieved under normoammonemic conditions (19). Hyperammonemia increases the astrocytic glutamine pool approximately threefold, and, consequently, the amount of label incorporated into brain metabolites is proportionately higher. Under hyperammonemic conditions the contribution of astrocytic glucose oxidation was found to be somewhat lower (4–7 % of the total oxidative metabolic rate) than that measured in human brain with [2-13 C]acetate. This difference may reﬂect either the different physiological conditions of these studies, or differences in the relative proportions of astrocytes and neurons in rat andhuman brain.

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It is thought that over 30 % of the synapses in the cerebral cortex may be GABAergic (40–42), and almost all of the brain GABA pool is localized to GABAergic neurons under normal conditions. GABA is synthesized from glutamate in GABAergic neurons by the enzyme glutamic acid decarboxylase (GAD). Thus, by following the ﬂux of 13 C label from [1-13 C]glucose into the GABA pool it is possible to obtain an estimate of oxidative glucose metabolism in the GABAergic neuron (10). The time course of label ﬂux into the GABA pool has been determined by in vitro MRS analysis of cortical extracts from rats infused with [1-13 C]glucose (43–45). Calculation of the relative rates of glucose oxidation in the glutamate and GABA pools suggests that the rate of glucose oxidation in GABAergic neurons is 10–20 % of total neuronal glucose oxidation (44). These ﬁndings and other related studies of GABA metabolism will be discussed in more detail in the following chapter. Thus, measurements of astrocyte and GABAergic neuron metabolism indicate that these cell populations account for approximately 15 % each of total cortical glucose consumption, leaving approximately 70 % for glutamatergic neurons. Once again, this is in good agreement with the ﬁndings from the [1-13 C]glucose studies and their comparison with measurements of total glucose utilization. Since glutamatergic pyramidal cells exhibit a high electrical activity and a large percentage of cortical synapses are glutamatergic (42, 46), it is perhaps not surprising that the greatest fraction of total glucose oxidation is associated with glutamatergic neurons.

5.2. MRS MEASUREMENTS OF NEUROTRANSMITTER FLUXES The metabolism of glutamatergic neurons, GABAergic neurons and astrocytes is coupled by neurotransmitter cycles. The concept of such a cycle between glutamatergic neurons and astrocytes was ﬁrst postulated in the early 1970s by van den Berg and colleagues (11), based on data from 14 C labeling studies (47, 48). The purpose of this cycle is to enable rapid removal of the neurotransmitter from the synaptic cleft, whilst preventing depletion of the nerve terminal glutamate pool by synaptic release. Although glutamate transporters have been found on both pre- and post-synaptic nerve terminals (see Bergles et al., 1999 (49) for review), a number of studies have now demonstrated that the majority of glutamate released from nerve terminals is taken up by surrounding astrocytes (50–52). Within astrocytes glutamate is converted to glutamine, by glutamine synthetase an exclusively glial enzyme (23). Glutamine is subsequently transported into the

Figure 5.2. Schematic diagram illustrating ﬂow of 13 C label from isotopic precursors into brain amino acids in the neuronal and astrocytic compartments. (A) [1-13 C]glucose is metabolized to [3-13 C]pyruvate by the glycolytic pathway, and label is subsequently transferred to the TCA cycle by the pyruvate dehydrogenase (PDH) pathway. Label is transferred via the TCA cycle to the C-4 of α-ketoglutarate (α-KG). The α-KG pool is in rapid exchange with the glutamate pool via the amino acid transaminases and mitochondrial/cytosolic transporters, and consequently glutamate is also labeled at C-4. Although these pathways are active in both neurons and astrocytes, the ﬂux of label from [1-13 C]glucose to [4-13 C]glutamate measured by 13 C MRS is taken to reﬂect primarily the neuronal compartment owing to the small size (and, hence, low sensitivity) of the astrocytic glutamate pool. (B) [2-13 C]acetate is metabolized to [2-13 C]acetyl CoA and enters the astrocytic TCA cycle. Label passes into the astrocytic glutamate pool at C-4 and, subsequently, into glutamine C-4 via glutamine synthetase (GS) where it is ﬁrst observed by 13 C MRS. Acetate is exclusively used in the brain by astrocytes and, consequently, this label ﬂux reﬂects metabolism solely within the astrocytic compartment. (C) [2-13 C]glucose metabolized via the astrocytic-speciﬁc pathway of pyruvate carboxylase (PC) brings label into the C-3 position of α-KG, and subsequently of the small astrocytic glutamate pool and glutamine (scrambling of label in the TCA cycle also results in C-2 enrichment of these metabolites). [2-13 C]glucose metabolized via the PDH pathway (in both neurons and astrocytes) results in labeling of acetyl CoA at C-1, which does not contribute to labeling in either the C-3 or C-2 positions of glutamate and glutamine. Consequently, as with [2-13 C]acetate, label ﬂux into these positions reﬂects metabolism solely within the astrocytic compartment.

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Figure 5.3. The glutamate–glutamine cycle. (A) Illustration of the glutamate–glutamine cycle between neurons and astrocytes. Glutamate (Glu) released from nerve terminals acts on a number of post-synaptic receptors, including ionotropic glutamate receptors, N-methyl-D-aspartate receptors and metabotropic glutamate receptors. Subsequently, this glutamate is taken up predominantly by surrounding astrocytes and converted to glutamine (Gln) by glutamine synthetase. Glutamine is then released for uptake by neurons, glutamate resynthesis via glutaminase, and re-packaging in vesicles. (B) Schematic diagram of the glutamate–glutamine cycle in the absence of any other contributing pathways to the glutamine synthetase ﬂux. Under these conditions the rate of glutamine synthesis (Vgln ) would equal the rate of glutamate–glutamine cycling (Vcycle ). (C) Schematic diagram of the glutamate–glutamine cycle illustrating the contribution of ammonia detoxiﬁcation to glutamine synthesis. Nitrogen inﬂux to the brain in the form of ammonia, at a rate Vtrans , is balanced by nitrogen efﬂux in glutamine, at a rate Vefﬂux . At the same time, loss of carbon skeletons from the brain in glutamine is balanced by net synthesis of carbon skeletons via the pyruvate carboxylase pathway in astrocytes, at a rate Vpc . Under these conditions, the total rate of glutamine synthesis can be deﬁned as the sum of anaplerotic glutamine synthesis and glutamate neurotransmitter cycling (Vgln = Vpc + Vcycle ).

extracellular space, taken up by neurons and converted back to glutamate by phosphate-activated glutaminase (53). Thus, a ‘glutamate–glutamine cycle’ between neurons and astrocytes is formed (Figure 5.3(A)). Similarly, it is now thought that GABA released into the synaptic cleft participates in a neuronal–astrocytic cycle, and this will be discussed in detail in the following chapter. Since the initial proposal of a glutamate–glutamine cycle between neurons and astrocytes, this concept has gained much support from a variety of in vitro studies, including enzyme localization studies, isotope labeling studies in cells and brain slices,

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and immunohistochemical studies of the cellular distribution of glutamate and glutamine (for review see Erecinska and Silver, 1990 (54)). However, prior to the development of in vivo 13 C MRS techniques there was no methodology available by which these cycles could be demonstrated in vivo. Furthermore, in vitro measurements of the glutamate–glutamine cycle were unable to determine the magnitude of this pathway. In this section, we will discuss how MRS can be used to measure neurotransmitter cycle ﬂuxes in vivo.

5.2.1. Measuring the Glutamate–Glutamine Cycle As described above, 13 C label from [1-13 C]glucose rapidly labels the large neuronal pool of glutamate. Thus, synaptic release of glutamate followed by uptake and conversion to glutamine in astrocytes, should result in label entering astrocytic glutamine via the glutamine synthetase pathway. Early 13 C MRS studies of human occipital/parietal cortex showed clearly that glutamine is labeled rapidly from [1-13 C]glucose in the human cerebral cortex (3, 7) (Figure 5.1). Based on these data, it was suggested that the 13 C MRS labeling time courses of glutamate and glutamine could be used to calculate the rate of glutamine synthesis, and from this the rate of the glutamate–glutamine cycle. The ﬁrst measurement of glutamine synthesis from 13 C MRS data acquired from human brain (3) was reported by Mason et al. in 1995 (7). At the time a relatively simplistic metabolic model was used to analyze the 13 C MRS data, and it was concluded that the measurement contained a high degree of uncertainty. In subsequent years much work has been done to reﬁne the metabolic model (see previous chapter) and to establish the validity of this measurement of glutamine synthesis as a measure of glutamate–glutamine cycling. A signiﬁcant caveat to this approach is that the relationship between glutamine synthesis and glutamate–glutamine cycling is dependent on the contribution of any other signiﬁcant ﬂuxes through the glutamine synthetase pathway in astrocytes, which could add to the labeling observed in glutamine. Previously, it has been suggested that glutamine synthesis acts primarily as a detoxiﬁcation pathway for blood-borne ammonia entering the brain (55) (Figure 5.3(C)). Consequently, in order to validate the measurements of glutamate–glutamine cycling the contribution of ammonia detoxiﬁcation to the total rate of glutamine synthesis must ﬁrst be determined. The contribution of ammonia detoxiﬁcation to glutamine synthesis will be determined by the rate of net ammonia transport into the brain, where this is considered to be the main source of ammonia to the brain. The issue of other sources of ammonia is reviewed in detail in Sibson et al., 1998 (56), and will not be discussed here. Nitrogen inﬂux to the brain in the form of ammonia is balanced by nitrogen efﬂux in glutamine (55) (Figure 5.3(C)). At the same time, loss of carbon skeletons from the brain in glutamine is balanced by net synthesis (anaplerosis) of carbon skeletons (Figure 5.3(C)), via the pyruvate carboxylase pathway in astrocytes (19, 56). Thus, the total rate of glutamine synthesis can be deﬁned as the sum of anaplerotic glutamine synthesis and glutamate neurotransmitter cycling. The contribution of ammonia detoxiﬁcation to total glutamine synthesis has been determined using 13 C MRS in rats at two different levels of plasma ammonia–normoammonemia (∼0.05 mM) and hyperammonemia (0.4–0.5 mM). Using [1-13 C]glucose as the isotopic precursor, the rate of glutamine synthesis was measured at the two levels of plasma ammonia and found to increase under hyperammonemic conditions. As described above, when [213 C]glucose is used as the isotopic precursor labeling in the internal positions of glutamine arises entirely as a consequence of ﬂux through the astrocytic pyruvate carboxylase pathway. Therefore, the rate of anaplerosis via this pathway can be determined from the ﬂux of label from [2-13 C]glucose into glutamine. By this method, under similar hyperammonemic conditions to the [1-13 C]glucose study, an increase in anaplerotic ﬂux was measured compared with normoammonemic conditions (19). This increase in anaplerotic ﬂux was found to be similar in magnitude to both the increase in glutamine synthesis and the increase in net ammonia inﬂux into the brain that has been measured by 15 N MRS, again under similar hyperammonemic conditions (57). During infusion of 15 N-labeled ammonia, 15 N MRS can be used to follow ﬁxation of the 15 N label in the cerebral pools of glutamate and glutamine, which reﬂects net ammonia transport into

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the brain. The good agreement of these ﬁndings lends strong support to the hypothesis that the contribution of ammonia detoxiﬁcation to glutamine synthesis is determined by the rate of net ammonia transport into the brain. These experiments demonstrated that even under hyperammonemic conditions the fraction of glutamine synthesis that is attributable to detoxiﬁcation is relatively small (∼30 %) compared to the contribution of glutamate–glutamine cycling (∼70 %). Moreover, under normoammonemic conditions the rate of ammonia transport into the brain can be calculated to be extremely low (55, 56). Consequently, the contribution of ammonia detoxiﬁcation to glutamine synthesis under normal physiological conditions is very small (<10%), and the measurement of glutamine synthesis largely reﬂects glutamate–glutamine cycling (17, 56). Thus, the studies described above provided initial support for the idea that the 13 C MRS approach, in combination with an infusion of [1-13 C]glucose, is a viable means of measuring the rate of glutamate–glutamine cycling in vivo. Nevertheless, once this principle was established it was important to validate the 13 C MRS measurements of the glutamate–glutamine cycle, and indeed the existence of such a cycle in vivo, since this single measurement is potentially open to a number of different interpretations. One of the disadvantages of using [1-13 C]glucose to measure the rate of the glutamate–glutamine cycle is that the 13 C label will enter both the astrocytic and neuronal glutamate pools directly via pyruvate dehydrogenase. Thus, labeling of the astrocytic glutamine pool could occur in the absence of any neuron–astrocyte cycling, and simply reﬂect exchange between glutamate and glutamine within astrocytes (Figure 5.4(A)). In this situation it would have to be argued that neuronal glutamate was replenished by pre-synaptic glutamate re-uptake. A third potential model of neurotransmitter metabolism that has been proposed in recent years is one based on cycling of α-ketoglutarate (α-KG) rather than glutamine from astrocytes to neurons (Figure 5.4(B)) (58). In this model glutamate taken up by astrocytes is converted to α-KG, by either oxidation or transamination, and this is then released for uptake and resynthesis of glutamate by neurons. With data obtained from a single experimental approach it is possible that a number of different metabolic models will equally well ﬁt those data, and it is not possible to reliably determine which model most accurately represents the true metabolic situation. Consequently, a number of different approaches have been employed in order to validate the 13 C MRS measurement of glutamate–glutamine cycling and to provide, thereby, support for the existence of such a cycle in vivo.

5.2.2. Validating the

13

C MRS Measurement of Glutamate–Glutamine Cycling

5.2.2.1. Correlation with Neuronal Activity One approach that has been used is to measure the rate of the cycle under conditions that alter neuronal glutamate neurotransmitter release. Neuronal glutamate release increases with increased action potential propagation and neuronal depolarization. Therefore, if the rate of 13 C ﬂux from glutamate into glutamine measured by 13 C MRS truly reﬂects synaptic glutamate release, and glutamate–glutamine cycling, then the calculated rate of this pathway should correlate with brain electrical activity. To test this prediction 13 C MRS was used to measure the rate of glutamine synthesis in the rat cerebral cortex at three levels of electrocortical activity; isoelectric EEG induced by high dose pentobarbital anesthesia, and two milder levels of anesthesia (56). Under isoelectric conditions, at which minimal glutamate release takes place, almost no glutamine synthesis was measured (0.04 µmol/min/g). Above isoelectricity, the rate of glutamine synthesis increased with higher electrical activity (Figure 5.5). These ﬁndings provide support for the hypothesis that the 13 C MRS measurement of glutamine synthesis primarily reﬂects glutamate–glutamine cycling rather than internal exchange of 13 C label within astrocytes, which would not be expected to change with the level of neuronal activity.

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(A) Pre-synaptic glutamatergic terminal

Astrocyte end-process Glu Gln Glu

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Post-synaptic glutamatergic terminal (B) Pre-synaptic glutamatergic terminal

Astrocyte end-process a-KG a-KG Glu

Glu

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Figure 5.4. Alternative models of glutamate metabolism. (A) Illustration of the ‘internal exchange’ model for glutamate (Glu) and glutamine (Gln) within astrocytes. In this situation neuronal glutamate would have to be replenished by presynaptic glutamate re-uptake. (B) Illustration of the proposed glutamate–α-ketoglutarate cycle between neurons and astrocytes. In this model glutamate taken up by astrocytes is converted to α-ketoglutarate (α-KG), by either oxidation or transamination, and this is then released for uptake and resynthesis of glutamate by neurons.

5.2.2.2. Alternative 13 C-labeled Precursors A second approach to validating the 13 C MRS measurement of glutamate–glutamine cycling is to use alternative isotopically labeled precursors, which enter the metabolic pathways of the brain speciﬁcally via different cell types and in particular astrocytes. If label is introduced speciﬁcally into astrocytic metabolite pools, then subsequent appearance of label in neuronal metabolites provides direct evidence of substrate trafﬁcking between these cells and could not be attributed to an exchange ﬂux within a single cell population. As discussed above, [2-13 C]glucose can be utilized as an astrocyte-speciﬁc precursor. Since the TCA cycle intermediate pools in astrocytes are very small, the isotopic label is ﬁrst observed in glutamine. In the [2-13 C]glucose experiment, if there is signiﬁcant cycling of labeled glutamine from the astrocyte to the neuron (as predicted by the glutamate–glutamine cycle), label would be expected to appear subsequently in neuronal glutamate. Labeling of neuronal glutamate has been demonstrated using [2-13 C]glucose in rat brain (19). Spectra acquired during [1-13 C]- and [2-13 C]glucose infusions, respectively, under similar physiological conditions demonstrate that in the [1-13 C]glucose experiment there is more rapid labeling of glutamate than glutamine, whilst in the [2-13 C]glucose experiment glutamine labeling precedes that of

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glutamate. This reversal of the precursor–product relationship can be seen clearly in the isotopic labeling curves from the two experiments shown in Figure 5.6(A,B). The appearance of label in the neuronal glutamate pool indicates that there is indeed substrate trafﬁcking between astrocytes and neurons, and that labeling of glutamine in the [1-13 C]glucose experiment does not simply reﬂect intra-astrocytic exchange of label between glutamate and glutamine. However, it does not differentiate between the different substrates that may participate in the astrocyte–neuron trafﬁcking. In order to determine which model (glutamate–glutamine cycling or glutamate–α-KG cycling) most accurately reﬂects the true metabolic situation, it is possible to apply these two different metabolic models to the data acquired from both the [1-13 C]glucose and [2-13 C]glucose experiments to see which model ﬁts both sets of data most accurately. The assumption is that, whilst an incorrect model could ﬁt one set of data reasonably well, it is unlikely to ﬁt two independent sets of data accurately. When both models were used to ﬁt the data from both [1-13 C]- and [2-13 C]glucose studies it was found that the glutamate–glutamine cycling model provided a consistently better ﬁt to both data sets (Figure 5.6(A,B)) than the glutamate–α-KG cycling model (Figure 5.6(C,D)) (19). These results indicate that the glutamate–glutamine cycle accounts for the majority of neuronal–astrocyte glutamate trafﬁcking, although contributions from alternative trafﬁcking pathways, such as the glutamate–α-KG cycle, cannot be excluded. Since, the [2-13 C]glucose experiment does not differentiate between label transferred from astrocyte to neuron in either glutamine or α-KG, the ﬂux of label into glutamate reﬂects the sum of all substrate trafﬁcking pathways from astrocytes to neurons. In contrast, in the [1-13 C]glucose experiment ﬂux of label from neuronal glutamate to astrocytic glutamine reﬂects glutamate–glutamine cycling alone. Consequently, comparison of the rates obtained in the two separate studies should indicate the fraction of substrate trafﬁcking from astrocytes to neurons that can be attributed to the glutamate–glutamine cycle. The absolute rates of cycling measured using the different isotopic precursors are in close agreement (0.20 ± 0.08 versus 0.20 ± 0.10 µmol/ min /g), indicating that, within the standard error of the measurement, the maximum potential contribution of the α-KG pathway to the total astrocyte–neuron cycling ﬂux is ∼25 %. These experiments were conducted under hyperammonemic conditions since, as explained in a previous section, labeling from [2-13 C]glucose is extremely low under

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Figure 5.6. Graphs demonstrating 13 C enrichment of cortical glutamate and glutamine in rat brain during infusion of different isotopic precursors, acquired under hyperammonaemic conditions (data from Sibson et al., 1997, 2001). The data clearly demonstrate reversal of the precursor–product relationship between glutamate and glutamine labeling. During infusion of [1-13 C]glucose (A), label ﬁrst appears in [4-13 C]glutamate ( ) and, subsequently, in [4-13 C]glutamine (). In contrast, during infusion of [2-13 C]glucose (B), label ﬁrst appears in [3-13 C]glutamine () and, subsequently, in [3-13 C]glutamate ( ). In (A) and (B) the solid lines indicate ﬁts to the data obtained via metabolic modeling using the glutamate–glutamine cycling model. In (C) and (D) the solid lines indicates ﬁts to the same data obtained using the glutamate–α-KG cycling model. It can be seen that the glutamate–glutamine cycling model provides a better ﬁt to both data sets than the glutamate–α-KG cycling model.

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normoammonemic conditions. Comparison of data from ex vivo [2-13 C]glucose experiments and in vivo [1-13 C]glucose experiments performed under normoammonemic conditions (19) also suggest that the α-KG pathway could contribute 25–30 % of the total cycling ﬂux. Very recently, experiments in humans infused with [2-13 C]acetate have been performed, and these studies have yielded results that are consistent with the [1-13 C]- and [2-13 C]glucose studies in rats. As with the [2-13 C]glucose experiment, clear differences in labeling patterns were observed when data acquired during [2-13 C]acetate infusion (34) were compared with those obtained from a [1-13 C]glucose experiment (2) (Figure 5.7). These ﬁndings are in accord with previous ﬁndings from animal studies (in vivo and ex vivo) (24, 30), indicating that acetate and glucose are metabolized primarily in different metabolic compartments – astrocytes and neurons, respectively. The observed ratio of glutamate/glutamine labeling (∼0.63) during [2-13 C]acetate infusion indicated that there is substantial transfer of 13 C label from astrocytes to neurons. The rate of substrate cycling obtained from this experiment (0.32 ± 0.07 µmol/ min /g) was again in close agreement with the rate of glutamate–glutamine cycling obtained from [1-13 C]glucose studies under identical conditions (0.32 ± 0.05 µmol/ min /g). Calculations suggest that, within the error of the 13 C measurements, a glutamate–α-KG cycle could account for 10 % of this substrate cycling (34).

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A third approach to validating the measurement of glutamate–glutamine cycling is to use 15 N MRS to measure glutamine synthesis through label incorporation from 15 N-labeled ammonia into the amide position of cerebral glutamine (57, 59–61). The rates of glutamine synthesis obtained in these studies have varied somewhat, but this can largely be explained by the fact that different metabolic models have been used to ﬁt the data. It has been shown that if the same metabolic model is used to ﬁt both 13 C and 15 N MRS data obtained under similar experimental conditions then the rates of glutamate–glutamine cycling obtained from both MRS studies agree closely (57). Furthermore, since 15 N-labeled ammonia is exclusively incorporated into cerebral metabolites in astrocytes, the same hypothesis holds as with the other astrocytespeciﬁc precursors – that label ﬂux from glutamine into the large, neuronal glutamate pool is indicative of astrocyte–neuron substrate cycling. In these studies, it is clear from the labeling patterns in glutamine and glutamate that this is, indeed, the case and that label appears ﬁrst in glutamine and only subsequently in glutamate (57, 59–61). It is extremely unlikely that a model will accurately ﬁt data obtained from several independent experimental approaches if it does not reasonably reﬂect the metabolic situation. The same metabolic model, involving a ﬂux of glutamate from neurons to astrocytes and a reciprocal ﬂux of glutamine from astrocytes to neurons, has now been shown to ﬁt data obtained using 13 C MRS and multiple different 13 C-labeled isotopic precursors ([1-13 C]glucose, [2-13 C]glucose and [2-13 C]acetate), as well as data obtained from 15 N MRS studies. These studies strongly support the existence of a glutamate–glutamine cycle between neurons and astrocytes in vivo, and have demonstrated that 13 C MRS may be used in vivo to measure the rate of glutamatergic neuronal activity, in the form of glutamate–glutamine cycling. The rate of the glutamate–glutamine cycle found in these studies of rat cerebral cortex is of a similar order of magnitude (0.20–0.40 µmol/ min /g) as the rate of oxidative glucose consumption measured by 13 C MRS. In addition, similar rates of glutamate–glutamine cycling (0.26–0.32 µmol/ min /g) have recently been measured in human occipital/parietal cortex (2, 4, 5) by the same methodology. These ﬁndings indicate that the glutamate–glutamine cycle is a major metabolic pathway in the mammalian cortex,

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and the predominant route by which neuronal glutamate pools are replenished. The rapid incorporation of 13 C label into glutamine via the glutamate–glutamine cycle indicates that the vesicular pool of glutamate in neurons turns over rapidly and is in dynamic equilibrium with cytosolic glutamate. This contradicts the long-standing view of functionally isolated transmitter and metabolic glutamate pools in neurons (27, 62). However, this concept had been established on the basis of in vitro studies of cell or tissue preparations, which exhibit little or no synaptic activity. In contrast, it has been shown more recently that inhibition of PAG (through which the neuronal glutamate pool is replenished) rapidly depletes both the synaptic and whole cell glutamate pools in rat cerebral cortex (13), in agreement with the 13 C MRS observations of a rapid vesicular turnover. Finally, these studies highlight the importance of astrocytes in maintaining synaptic glutamate homeostasis, which has long been a source of controversy.

5.3. ENERGETIC REQUIREMENTS OF NEUROTRANSMISSION Neuronal activity requires energy which is provided almost exclusively by oxidation of glucose (1). Since the brain has almost no energy reserve, a continuous vascular supply of glucose and oxygen is mandatory to sustain neuronal activity. This supply is regulated locally and dynamically to meet the increased energetic demand of functional activation. Functional imaging utilizes this association between increased neuronal activity and energetic demand, by measuring changes in either blood ﬂow, glucose utilization or oxygen metabolism and delivery (63). The implicit assumption of functional imaging methodologies is that changes in one or more of these parameters accurately reﬂect changes in neuronal activity. Despite the increasing use of these imaging tools in both basic and clinical applications, the neurobiological processes responsible for the imaging signals measured are still unknown. Recent advances in the ﬁeld suggest that in some brain regions glutamate release may trigger both vascular and metabolic responses via different signaling processes (for review see Bonvento et al., 2002 (64)). Thus, glutamate may be a key co-coordinator of the physiological responses underlying the signal changes observed in brain imaging. 13 C MRS has provided a means by which the relationship between glutamatergic neurotransmission and energy metabolism can be tested in vivo. This has been achieved in the rat brain by using 13 C MRS to simultaneously measure the rates of glutamate–glutamine cycling and neuronal glucose oxidation in the cerebral cortex at different levels of neuronal activity (18). As described in the previous section, electrocortical activity was varied using differing levels of anesthesia, and when neuronal glucose oxidation was plotted against the rate of glutamate–glutamine cycling a linear relationship was obtained (Figure 5.8(A)). The observed relationship between oxidative glucose metabolism and glutamate–glutamine cycling demonstrates two important points – ﬁrstly, that there is a molar (1:1) stoichiometry between these two rates above a basal level of energy metabolism (isoelectric EEG), and secondly, that the basal level of glucose consumption (i.e. unrelated to synaptic activity) is only 15–20 % of resting levels. The ﬁrst of these points implies that the energy requirements of pre- and post-synaptic events related to neuronal ﬁring and synaptic transmission may be met by the oxidative metabolism of 1 mol of glucose for every mole of glutamate released synaptically. These studies are not able to assign fractions of energy utilization to each of the individual processes involved (action potential propagation, restoration and maintenance of membrane potentials, vesicle recycling and glutamate cycling), although recent theoretical calculations have begun to address this issue (65). This analysis (based on a mean neuronal action potential frequency of 4 Hz) has shown that action potentials are responsible for a large fraction (47 %) of total ATP consumption, as restoration of the sodium and potassium gradients occurs via the Na+ /K+ -ATPase. Increased activity of the Na+ /K+ -ATPase is also required to reverse ion movements evoked by the activation of post-synaptic

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Figure 5.8. Graphs illustrating the relationship between oxidative glucose consumption and glutamate–glutamine cycling in the cerebral cortex. (A) 13 C MRS data acquired from rat cerebral cortex at different levels of neuronal activity yielded rates of oxidative glucose consumption and glutamate–glutamine cycling (data from Sibson et al., 1998a). Three different levels of neuronal activity were achieved through the use of graded anesthesia: isoelectric EEG (); moderate anesthesia ( ); and mild anesthesia (). It should be noted that the units of the x and y axes are the same and, hence, these two metabolic rates increase with a 1:1 molar stoichiometry above a basal level of energy consumption. The basal level of energy metabolism was measured at isoelectric EEG (i.e. no neurotransmission or glutamate–glutamine cycling) and, thus, reﬂects the energetic requirements of the brain that are unrelated to neuronal activity (e.g. macromolecule turnover, maintenance of membrane structure and axoplasmic transport). (B) The same data as shown in (A) are superimposed on 13 C MRS data obtained from human cerebral cortex ( ). Data are from Shen et al., 1999 and Gruetter et al., 2001 (mean ± S.D. for each metabolic rate). This graph demonstrates that the measurements of oxidative glucose consumption and glutamate–glutamine cycling obtained from human cerebral cortex are entirely consistent with the rates obtained in the rat cerebral cortex. The human cortical rates appear to lie at the lower end of those obtained in the rat cortex, corresponding to a state of moderate anesthesia in the rat. The reasons for this are possibly twofold – ﬁrstly, that the human cerebral cortex has a threefold to tenfold lower density of neurons and, secondly, that in the human measurements the volume of tissue under observation contains both grey and white matter. As yet, an analogous 13 C MRS study of human brain under different levels of neuronal activity has not been performed. Consequently, it remains to be seen whether a linear relationship between neurotransmission and energy metabolism exists in man.

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NMDA and non-NMDA ionotropic glutamate receptors, and this activity has been calculated to account for as much as 34 % of total ATP usage. Therefore, the prevailing hypothesis is still that the majority (∼80 %) of total cerebral energy consumption subserves restoration of the ionic gradients and resting membrane potentials that are modiﬁed during the excitation phase of neuronal activation (65). These calculations can also be expressed in terms of the pre-synaptic, post-synaptic and glial energy requirements as shown in Figure 5.9. The second ﬁnding, of a low basal metabolic rate measured under isoelectric conditions, is in close agreement with the recent theoretical analysis of the energetic expenditure of the brain (65), and presumably supports basic cellular processes, unrelated to signaling, such as macromolecule turnover (proteins, lipids, oligonucleotides), maintenance of membrane structure, and axoplasmic transport. However, this represents a departure from the long-held view that the majority of energy consumption in the brain subserves these socalled ‘housekeeping’ functions, rather than those involved in neuronal activity and synaptic transmission.

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Figure 5.9. Glutamatergic neurotransmission involves a number of energy-requiring steps. Initially, propagation of an action potential along an axon to a pre-synaptic terminal results in changes in ionic gradients across the axonal membrane. Restoration of these ion gradients requires the activity of Na+ /K+ -ATPase pumps. Next, glutamate release is triggered by Ca2+ inﬂux into the pre-synaptic terminal, which must be pumped out at the expense of ATP. Glutamate released into the synaptic cleft acts on several different post-synaptic receptors (iGluR, ionotropic glutamate receptor; NMDAR, N-methyl-D-aspartate receptor; mGluR, metabotropic glutamate receptor), and also on presynaptic and glial receptors (not shown). Activation of these receptors causes ion movements (mostly Na+ ) and second messenger cascades, and re-establishment of ion gradients is performed by the Na+ /K+ -ATPase. If enough depolarization of the post-synaptic neuron is produced, an action potential will ensue, and, once again, restoration of the ion gradients occurs via the Na+ /K+ -ATPase. Finally, glutamate recycling involves Na+ -dependent uptake by astrocytes and conversion to glutamine, as well as vesicle recycling in neurons, all of which consume ATP. Recent evidence suggests that astrocytes may release glutamate that participates in neuronal depolarization, although the extent of this astrocytic release remains to be determined. It has recently been calculated that in terms of the pre-synaptic, post-synaptic and glial processes, these energy requirements can be broken down to give ∼42 % pre-synaptic consumption (axonal action potentials, pre-synaptic Ca2+ entry and vesicularization of glutamate), 53 % post-synaptic consumption (glutamate receptor activation, dendritic and somatic action potentials and post-synaptic resting potentials) and 5 % glial consumption (resting potentials, glutamate uptake and glutamine synthesis).

On reﬂection, however, it is perhaps not surprising that neuronal signaling dominates the energetic cost of the brain, given that its primary functions are information processing and signaling.

5.3.1. Glutamate as a Mediator of Neurometabolic Coupling The demonstrated stoichiometric relationship between oxidative glucose consumption and glutamate–glutamine cycling is supportive of the concept that glutamate release may be a means by which energy metabolism can be regulated in line with neuronal activity. This hypothesis was proposed several years ago by Pellerin and Magistretti (66) based on in vitro studies of cultured astrocytes. Those experiments demonstrated that glutamate induced increases in both glucose utilization and lactate production by astrocytes. These effects on glucose metabolism were not receptor mediated, but rather dependent on glutamate transport. As glutamate transport is coupled to the sodium ion gradient with a precise stoichiometry of three sodium ions for each transported glutamate molecule, glutamate uptake leads to a massive entry of sodium

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ions into astrocytes (as demonstrated by ﬂuorescence microscopy) (67). Further studies in cultured astrocytes have shown that glutamate enhances the activity of a speciﬁc glial Na+ /K+ -ATPase subunit (68), and that ouabain, a speciﬁc inhibitor of the Na+ /K+ -ATPase, prevents the enhancement of glucose utilization and lactate production by glutamate (66). Thus, the activity of this glial enzyme has been directly implicated in the metabolic response. The concept that glial glutamate transporters may play a role in neurometabolic coupling has recently received support from in vivo studies in glutamate transporter (GLAST and GLT-1) knockout mice (69). In these studies the metabolic response to somatosensory activation was signiﬁcantly decreased compared to wild type mice, as demonstrated by 2DG autoradiography. In a second study, glucose utilization in rat brain was also found to be decreased following the downregulation of the astrocytic glutamate transporter GLAST by antisense oligonucleotides (70). Thus, these ﬁndings suggest an attractive mechanism of coupling energy metabolism to neuronal activity via astrocytic glutamate uptake, which is supported by the relationship between glucose oxidation and glutamate-glutamine cycling obtained by 13 C MRS. It is important to note that, although astrocytic glutamate uptake could trigger at least part of the observed changes in glucose metabolism, the energetic requirements of astrocytic glutamate uptake and recycling are small. Approximately 5 % of total energy consumption is required to support glutamate uptake and recycling (65) (Figure 5.9), a percentage that would be consistent with the process being predominantly fuelled by glycolysis. This suggestion is supported to some extent by an ex vivo 13 C MRS study of mice in which the astrocytic TCA cycle was inhibited with ﬂuoroacetate (71). In this study, it was found that glutamate–glutamine cycling (as determined by 13 C labeling of glutamine from [1-13 C]glucose) was maintained to a substantial degree despite an almost complete inhibition of astrocytic oxidative metabolism. One might hypothesize, therefore, that glycolytic energy might support the very rapid process (∼1 ms (72, 73)) of glutamate uptake by astrocytes, whilst oxidative metabolism could support those processes of synaptic activity that can occur over a longer timeframe (e.g. restoration of membrane potentials and ionic gradients, recycling of vesicles). How this last aspect is regulated and whether glucose alone or other substrates (e.g. lactate) are oxidized by active neurons remain to be determined.

5.3.2. Implications for Studies of Human Brain Importantly, measurements of these metabolic rates in human cerebral cortex during infusion of either [1-13 C]glucose (2, 5) or, very recently, [2-13 C]acetate (34) are entirely consistent with the rates obtained in the rat cerebral cortex (Figure 5.8(B)). Interestingly, the human cortical rates lie at the lower end of those obtained in the rat cortex, which corresponds to a state of moderate anesthesia. The reasons for this are possibly twofold – ﬁrstly, that the human cerebral cortex has a threefold to tenfold lower density of neurons (74) and, secondly, that in the human measurements the volume of tissue under observation will contain both grey and white matter. In the ﬁrst case, although the density of synapses in human cortex is no different to that in rat cortex, Attwell and Laughlin (65) calculated that a tenfold lower density of neurons per se would result in a 54 % reduction in speciﬁc energy usage and oxygen consumption. In accord with this prediction, metabolic rates in neocortical grey matter of primate brain are, on average, ∼50 % lower than in rat (75–77). With respect to the second point, we would expect the rate of glutamate–glutamine cycling to be considerably higher in grey matter, which contains large numbers of synaptic terminals, than in white matter consisting predominantly of axon bundles. Correspondingly, white matter has been shown to have a considerably lower oxidative metabolic rate than grey matter (22). Consequently, the partial volume effect of including signiﬁcant fractions of white matter in the volume of tissue from which the 13 C measurement is made will signiﬁcantly reduce the measured rates of both cycling and glucose utilization. However, it should be noted that to date an analogous 13 C MRS study of human brain under different levels of neuronal activity has not been performed. Thus, it is not yet possible to determine whether a

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linear relationship between neurotransmission and energy metabolism, similar to that found in rat cerebral cortex, exists in man.

5.4. SUMMARY The application of MRS to the study of neurotransmitter and energy metabolism in the brain has provided several new insights into the relationship between brain metabolism and function. Glutamate release and recycling has been shown to be a major metabolic pathway in the cerebral cortex. Consequently, steady state measurements of neurotransmitter levels may reﬂect alterations in regional brain activity through feedback between neurotransmitter release and resynthesis. The glutamate–glutamine cycle appears to be the predominant route by which the neurotransmitter glutamate pool in neurons is replenished, and demonstrates the importance of astrocytes in maintaining synaptic glutamate homeostasis. Interestingly, the glutamate–glutamine cycle constitutes a signiﬁcant metabolic ﬂux in both resting, awake human brain and anesthetized rat brain, indicating that there is considerable neuronal activity even under ‘nonstimulated’ conditions. The 1:1 coupling between neurotransmission and neuroenergetics (above isoelectricity) provides a link between functional imaging and speciﬁc neuronal processes. This ﬁnding has implications for the interpretation of functional imaging signal changes, which may be most readily understood by converting the measured rates of glucose oxidation to fractions of total glucose consumption in the brain. In doing so, we ﬁnd that glucose oxidation by glutamatergic neurons comprises approximately 70 % of the total energy consumption of the brain, while the astrocytic and GABAergic neuron consumptions are estimated to be approximately 15 % each. Thus, the energy requirements of glutamatergic neurons potentially explain a large fraction of the glucose consumption of the resting awake human brain. Further, these ﬁndings may provide a mechanism for converting energy changes measured in human brain activation studies into a speciﬁc neuronal process, such as excitatory glutamate release. Should an analogous study of glutamate–glutamine cycling and oxidative glucose consumption over a range brain activity in human cortex yield a similar relationship to that found in rat cortex, then this will bridge the gap that currently exists between functional activation data (based on metabolic indices) and neuronal activity. Thus, establishing the functional imaging modalities, which have the advantages of greater spatial and temporal resolution than the 13 C MRS measurement, as true indices of neuronal activity.

REFERENCES 1 Siesjo B. Brain Energy Metabolism. New York: John Wiley and Sons Inc., 1978 2 Shen J, Petersen KF, Behar KL, Brown P, Nixon TW, Mason GF, Petroff OA, Shulman GI, Shulman RG, Rothman DL. Determination of the rate of the glutamate/glutamine cycle in the human brain by in vivo 13 C NMR. Proc Natl Acad Sci U S A 1999; 96: 8235–40 3 Gruetter R, Novotny EJ, Boulware SD, Mason GF, Rothman DL, Shulman GI, Prichard JW, Shulman RG. Localized 13 C NMR spectroscopy in the human brain of amino acid labeling from D-[1-13 C]glucose. J Neurochem 1994; 63: 1377–85 4 Gruetter R, Seaquist ER, Kim S, Ugurbil K. Localized in vivo 13 C-NMR of glutamate metabolism in the human brain: initial results at 4 tesla. Dev Neurosci 1998; 20: 380–8 5 Gruetter R, Seaquist ER, Ugurbil K. A mathematical model of compartmentalized neurotransmitter metabolism in the human brain. Am J Physiol Endocrinol Metab 2001; 281: E100–12. 6 Fitzpatrick SM, Hetherington HP, Behar KL, Shulman RG. The ﬂux from glucose to glutamate in the rat brain in vivo as determined by 1 H-observed, 13 C-edited NMR spectroscopy. J Cereb Blood Flow Metab 1990; 10: 170–9.

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7 Mason GF, Gruetter R, Rothman DL, Behar KL, Shulman RG, Novotny EJ. Simultaneous determination of the rates of the TCA cycle, glucose utilization, alpha-ketoglutarate/glutamate exchange, and glutamine synthesis in human brain by NMR. J Cereb Blood Flow Metab 1995; 15: 12–25 8 Mason GF, Pan JW, Chu WJ, Newcomer BR, Zhang Y, Orr R, Hetherington HP. Measurement of the tricarboxylic acid cycle rate in human grey and white matter in vivo by 1 H-[13 C] magnetic resonance spectroscopy at 4.1 T. J Cereb Blood Flow Metab 1999; 19: 1179–88. 9 Mason GF, Rothman DL, Behar KL, Shulman RG. NMR determination of the TCA cycle rate and alpha-ketoglutarate/glutamate exchange rate in rat brain. J Cereb Blood Flow Metab 1992; 12: 434–47. 10 Rothman DL, Sibson NR, Hyder F, Shen J, Behar KL, Shulman RG. in vivo nuclear magnetic resonance spectroscopy studies of the relationship between the glutamate–glutamine neurotransmitter cycle and functional neuroenergetics. Philos Trans R Soc London Ser B Biol Sci 1999; 354: 1165–77 11 van den Berg CJ, Garﬁnkel D. A stimulation study of brain compartments. Metabolism of glutamate and related substances in mouse brain. Biochem J 1971; 123: 211–8. 12 Ottersen OP, Zhang N, Walberg F. Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum. Neuroscience 1992; 46: 519–34 13 Conti F, Minelli A. Glutamate immunoreactivity in rat cerebral cortex is reversibly abolished by 6-diazo-5-oxo-Lnorleucine (DON), an inhibitor of phosphate-activated glutaminase. J Histochem Cytochem 1994; 42: 717–26. 14 Hyder F, Rothman DL, Mason GF, Rangarajan A, Behar KL, Shulman RG. Oxidative glucose metabolism in rat brain during single forepaw stimulation: a spatially localized 1 H[13 C] nuclear magnetic resonance study. J Cereb Blood Flow Metab 1997; 17: 1040–7. 15 Hyder F, Chase JR, Behar KL, Mason GF, Siddeek M, Rothman DL, Shulman RG. Increased tricarboxylic acid cycle ﬂux in rat brain during forepaw stimulation detected with 1 H[13 C]NMR. Proc Natl Acad Sci U S A 1996; 93: 7612–7. 16 Hyder F, Renken R, Rothman DL. In vivo carbon-edited detection with proton echoplanar spectroscopic imaging (ICED PEPSI): [3,4-(13)CH(2)]glutamate/glutamine tomography in rat brain. Magn Reson Med 1999; 42: 997–1003. 17 Sibson NR, Dhankhar A, Mason GF, Behar KL, Rothman DL, Shulman RG. In vivo 13 C NMR measurements of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling. Proc Natl Acad Sci U S A 1997; 94: 2699–704 18 Sibson NR, Dhankhar A, Mason GF, Rothman DL, Behar KL, Shulman RG. Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proc Natl Acad Sci U S A 1998; 95: 316–21 19 Sibson NR, Mason GF, Shen J, Cline GW, Herskovits AZ, Wall JE, Behar KL, Rothman DL, Shulman RG. in vivo (13)C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle ﬂux in rat brain during [2-13C]glucose infusion. J Neurochem 2001; 76: 975–89. 20 van Zijl PC, Chesnick AS, DesPres D, Moonen CT, Ruiz-Cabello J, van Gelderen P. in vivo proton spectroscopy and spectroscopic imaging of [1–13C]-glucose and its metabolic products. Magn Reson Med 1993; 30: 544–51. 21 Van Zijl PC, Davis D, Eleff SM, Moonen CT, Parker RJ, Strong JM. Determination of cerebral glucose transport and metabolic kinetics by dynamic MR spectroscopy. Am J Physiol 1997; 273: E1216–27. 22 Pan JW, Stein DT, Telang F, Lee JH, Shen J, Brown P, Cline G, Mason GF, Shulman GI, Rothman DL, Hetherington HP. Spectroscopic imaging of glutamate C4 turnover in human brain. Magn Reson Med 2000; 44: 673–9. 23 Martinez-Hernandez A, Bell KP, Norenberg MD. Glutamine synthetase: glial localization in brain. Science 1977; 195: 1356–8. 24 Bachelard H. Landmarks in the application of 13 C-magnetic resonance spectroscopy to studies of neuronal/glial relationships. Dev Neurosci 1998; 20: 277–88 25 Cruz F, Cerdan S. Quantitative 13C NMR studies of metabolic compartmentation in the adult mammalian brain. NMR Biomed 1999; 12: 451–62. 26 Bachelard H, Morris P, Taylor A, Thatcher N. High-ﬁeld MRS studies in brain slices. Magn Reson Imaging 1995; 13: 1223–6 27 Badar-Goffer RS, Ben-Yoseph O, Bachelard HS, Morris PG. Neuronal–glial metabolism under depolarizing conditions, A 13 C-n.m.r. study. Biochem J 1992; 282: 225–30.

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51 Bergles DE, Diamond JS, Jahr CE. Clearance of glutamate inside the synapse and beyond. Curr Opin Neurobiol 1999; 9: 293–8. 52 Rothstein JD, Dykes-hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, SChielke JP, Welty DF. Knockout of glutamate transporters reveals a major role for astroglial transport in excit otoxicity and clearance of glutamate. Neuron 1996; 16: 675–686 53 Kvamme E, Torgner IA, Svenneby G. Glutaminase from mammalian tissues. Methods Enzymol 1985; 113: 241–56 54 Erecinska M, Silver IA. Metabolism and role of glutamate in mammalian brain. Prog Neurobiol 1990; 35: 245–296 55 Cooper AJL, Plum F. Biochemistry and physiology of brain ammonia. Physiological 1987; 67: 440–519 56 Sibson NR, Shen J, Mason GF, Rothman DL, Behar KL, Shulman RG. Functional energy metabolism: in vivo 13 C-NMR spectroscopy evidence for coupling of cerebral glucose consumption and glutamatergic neuronalactivity. Dev Neurosci 1998; 20: 321–30 57 Shen J, Sibson NR, Cline G, Behar KL, Rothman DL, Shulman RG. 15N-NMR spectroscopy studies of ammonia transport and glutamine synthesis in the hyperammonemic rat brain. Dev Neurosci 1998; 20: 434–43 58 Shank RP, Campbell GL. Alpha-ketoglutarate and malate uptake and metabolism by synaptosomes: further evidence for an astrocyte-to-neuron metabolic shuttle. J Neurochem 1984; 42: 1153–61 59 Kanamori K, Ross BD. 15N n.m.r. measurement of the in vivo rate of glutamine synthesis and utilization at steady state in the brain of the hyperammonaemic rat. Biochem J 1993; 293: 461–8. 60 Kanamori K, Parivar F, Ross BD. A 15N NMR study of in vivo cerebral glutamine synthesis in hyperammonemic rats. NMR Biomed 1993; 6: 21–6. 61 Kanamori K, Ross BD, Chung JC, Kuo EL. Severity of hyperammonemic encephalopathy correlates with brain ammonia level and saturation of glutamine synthetase in vivo. J Neurochem 1996; 67: 1584–94. 62 Nicholls D, Attwell D. The release and uptake of excitatory amino acids. Trends Pharmacol Sci 1990; 11: 462–8 63 Raichle ME. Behind the scenes of functional brain imaging: a historical and physiological perspective. Proc Natl Acad Sci U S A 1998; 95: 765–72 64 Bonvento G, Sibson N, Pellerin L. Does glutamate image your thoughts? Trends Neurosci 2002; 25: 359–64 65 Attwell D, Laughlin SB. An energy budget for signaling in the grey matter of the brain. J Cereb Blood Flow Metab 2001; 21: 1133–45 66 Pellerin L, Magistretti PJ. Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci U S A 1994; 91: 10625–9 67 Chatton JY, Marquet P, Magistretti PJ. A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics. Eur J Neurosci 2000; 12: 3843–53 68 Pellerin L, Stolz M, Sorg O, Martin JL, Deschepper CF, Magistretti PJ. Regulation of energy metabolism by neurotransmitters in astrocytes in primary culture and in an immortalized cell line. Glia 1997; 21: 74–83 69 Voutsinos-Porche B, Bonvento G, Tanaka K, Magistretti P, Pellerin L. Evidence from knockout mice for a major role of the glial glutamate transporters GLAST and GLT-1 in neurometabolic coupling. J Cereb Blood Flow Metab 2001; 21 (Suppl. 1): S115 70 Cholet N, Pellerin L, Welker E, Lacombe P, Seylaz J, Magistretti P, Bonvento G. Local injection of antisense oligonucleotides targeted to the glial glutamate transporter GLAST decreases the metabolic response to somatosensory activation. J Cereb Blood Flow Metab 2001; 21: 404–12 71 Hassel B, Bachelard H, Jones P, Fonnum F, Sonnewald U. Trafﬁcking of amino acids between neurons and glia in vivo. Effects of inhibition of glial metabolism by ﬂuoroacetate. J Cereb Blood Flow Metab 1997; 17: 1230–8. 72 Diamond JS, Jahr CE. Transporters buffer synaptically released glutamate on a submillisecond time scale. J Neurosci 1997; 17: 4672–87 73 Clements JD. Transmitter timecourse in the synaptic cleft: its role in central synaptic function. Trends Neurosci 1996; 19: 163–71 74 Abeles M. Corticonics: Neural Circuits of the Cerebral Cortex. Cambridge: Cambridge University Press, 1991 75 Clarke J, Sokoloff L. Circulation and energy metabolism of the brain. In: Siegel G, Agranoff B, Albers R, Fisher S, Uhler M (eds), Basic Neurochemistry, 6th edn. Philadelphia: Lippincott-Raven, 1999; 637–669

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76 Kennedy C, Sakurada O, Shinohara M, Jehle J, Sokoloff L. Local cerebral glucose utilization in the normal conscious macaque monkey. Ann Neurol 1978; 4: 293–301 77 Sokoloff L, Reivich M, Kennedy C, Des Rosiers MH, Patlak CS, Pettigrew KD, Sakurada O, Shinohara M. The [14 C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem 1977; 28: 897–916

6 NMR Studies of the Metabolism and Energetics of GABA Neurotransmitter Pathways Kevin L. Behar Department of Psychiatry, Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

Douglas L. Rothman Department of Diagnostic Radiology, Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

6.1 Introduction 6.2 Neuronal Localization of GABA 6.2.1 In vivo Detection of GABA using NMR 6.3 Pathways of Brain GABA Synthesis and Catabolism 6.3.1 GABA Metabolism in the GABA Shunt 6.3.2 Glutamate Decarboxylase and GABA Synthesis 6.3.3 Alternate Pathways of GABA Synthesis 6.4 Role of GABA and Glutamine Transporters in GABA Metabolism 6.4.1 GABA Transporters 6.4.2 Glutamine Transporters 6.5 NMR Studies of GABAergic Function 6.5.1 Measurements of GABA Synthesis 6.5.2 GABA/Glutamine Cycling 6.6 NMR Studies of GABA Synthesis Regulation In vivo 6.7 Conclusions

Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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6.1. INTRODUCTION Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mature central nervous system. In the cortex, GABA may constitute as many as one-quarter of all synapses (Iversen and Schon, 1973), although such estimates are only approximate. GABA inhibition in the mature CNS involves both fast, point-to-point signaling on a timescale of milliseconds and slower tonic responses lasting seconds or longer. These responses are mediated mainly through chloride-dependent, GABAA receptors and potassiumdependent, G-protein linked, GABAB receptors, respectively (Simeone et al., 2003; Owens and Kriegstein, 2002). These different GABA responses may reﬂect functionally distinct intracellular pools of GABA and different release mechanisms. GABA is a product of glucose metabolism at the level of the tricarboxylic acid (TCA) cycle. GABA is synthesized from glutamate and oxidized to succinic acid in a metabolic ‘shunt’ that bypasses two enzymes of the TCA cycle. Whereas the enzymes of GABA synthesis are almost exclusively conﬁned to GABAergic neurons, the enzymes of GABA catabolism are more widespread, being found in astroglia as well as neurons. GABA released from neurons is removed both by reuptake and also by astroglia through speciﬁc GABA transporters (GATs). GABA removed by astroglia must be re-supplied because mature neurons do not possess the anaplerotic enzymes (e.g., pyruvate carboxylase) needed to catalyze carbon ﬁxation and de novo glutamate synthesis. In the cerebral cortex glutamine produced in astroglia provides a major fraction of the glutamate used in the re-synthesis of neurotransmitter glutamate and GABA. The cyclic ﬂow of these neurotransmitters from their respective neurons to astroglial glutamine and back again gives rise to glutamate/glutamine and GABA/glutamine cycles (Figure 6.1). The existence of carbon cycles involving GABA, glutamate, and glutamine has been recognized for over 30 years although the magnitude of their respective ﬂows in relation to glucose metabolism and energetics was not well known until recently. New insights into the energetics of the GABA and glutamate neurotransmitter pathways in relation to glucose metabolism are being revealed through the tools of modern neuroimaging and NMR spectroscopy. Developments in spectroscopy and labeling techniques along with comprehensive models of metabolic networks to analyze the dynamic isotopic labeling time courses of glutamate and glutamine have permitted the pathways of glucose metabolism and glutamate/glutamine cycle to be measured. These studies have revealed that glutamatergic neuronal activity accounts for a major fraction of glucose oxidation in the cortex and is coupled to neuronal glucose oxidation in a ∼ 1:1 relationship (Sibson et al., 1998; Rothman et al, 1999). The earliest experiments using [1-13 C]glucose could not distinguish the individual contributions made by glutamate and GABA to total cycling. More recently the challenge has been to separately quantify the ﬂuxes of these neurotransmitter pathways using astroglial targeted 13 C-labeled substrates (e.g., [213 C]glucose or [2-13 C]acetate) as described later in this chapter (also see Chapter 5). These studies have revealed that the GABA/glutamine cycle provides ∼1/5 of the total cycling measured using [1-13 C]glucose in the anesthetized rat cortex (Patel et al., 2003). In this chapter we brieﬂy describe the major and minor metabolic pathways of GABA synthesis and catabolism, GABA transport, and the role of astroglia in the GABA/glutamine cycle. The ﬂuxes of major and minor pathways of GABA metabolism are provided and discussed in the context of rate measurements using 13 C-labeled isotopes. The important role of glutamate decarboxylase in the regulation of GABA synthesis is discussed along with recent attempts using in vivo NMR to determine the contribution of the two brain isoforms of GAD to GABA synthesis. For a more comprehensive discussion of the NMR literature of GABA and metabolic compartmentation interested readers are encouraged to consult reviews of this subject by Bachelard (1998), Cruz and Cerdan (1999), and Waagepetersen et al. (1999).

6.2. NEURONAL LOCALIZATION OF GABA GABA is present in brain tissue at a concentration of about 2–5 µmol/g wet tissue weight and a large majority of this is concentrated in GABAergic neurons. In cerebellar Purkinje neurons the cellular concentration of

NMR Studies of the Metabolism and Energetics of GABA Neurotransmitter Pathways Neuron (Glutamatergic or GABAergic) Ac-CoA

Astrocyte Gln

Vcycle(tot)

Gln

Vgln a-KG

101

Glu

Ac-CoA

Glu a-KG

VGlu/Gln TCAn

TCAa Suc

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Suc

Figure 6.1. Schematic depiction of glutamate/glutamine and GABA/glutamine substrate cycles interconnecting glutamatergic and GABAergic neurons to astroglia. In astroglia GABA is readily metabolized through transamination and oxidation to succinic acid (Suc), which enters the astroglial TCA cycle. During transamination of GABA, α-ketoglutarate (α-KG) is converted to glutamate, which may then proceed to the formation of glutamine by glutamine synthetase. Thus, net uptake of GABA into astrocytes can lead indirectly to net glutamine formation, a process that requires a molar equivalent consumption of glucose-derived pyruvate or acetyl group donors to replace α-KG consumed in the synthesis of glutamine. The carbon skeleton of GABA entering astroglia in the GABA/glutamine cycle does not contribute directly to glutamine, unlike glutamate in the glutamate/glutamine cycle. The syntheses of glutamate and GABA are depicted with a single TCA cycle to emphasize net carbon ﬂows although glutamatergic and GABAergic neurons possess separate TCA cycles. Fluxes are depicted as follows: VGlu/Gln , glutamate/glutamine cycle ﬂux; VGABA/Gln , GABA/glutamine cycle ﬂux; Vcycle(tot) , total (Glu + GABA) cycling ﬂux; VGAD , rate of GABA synthesis; Vgln , rate of glutamine synthesis. TCAn and TCAa , neuronal and astroglial tricarboxylic acid cycle ﬂuxes, respectively. NMR measurements of neurotransmitter cycling using [1-13 C]glucose reﬂects total (glutamate + GABA) neurotransmitter cycling. The two cycles can be separately determined using astroglia targeted 13 C-labeled substrates (e.g., [2-13 C]glucose or [2-13 C]acetate).

GABA may reach 50–100 mM (Fonnum and Walberg, 1973). Based on studies of [3 H]GABA uptake, the number of GABA nerve terminals has been estimated to range from 14 to 51 % of all terminals depending on brain region (Iversen and Schon, 1973), although actual numbers may be less because GABA transporters are expressed on glia as well as neurons. GABA neurons also tend to be smaller in size than the glutamatergic principal neurons. From a metabolic and neuroimaging viewpoint, the fractional volume of tissue occupied by GABAergic nerve terminals is of greater signiﬁcance than their numbers in computations of metabolic rates. GABA and the enzyme catalyzing its synthesis, glutamate decarboxylase (GAD), are localized to GABAergic neurons although not exclusively. Excitatory granule and mossy ﬁber neurons in the dentate gyrus of the hippocampus, which use glutamate as neurotransmitter, also contain GABA and GAD and their synthesis is enhanced by increasing electrical activity (Sloviter et al., 1996; Cao et al., 1996; Gutierrez, 2002). GABA neurons are heterogeneous as a group in that many, if not all, contain one or more neuroactive peptides, such as VIP, parvalbumin, and calbindin. The concentration of GABA in interstitial ﬂuid is maintained at a very low level (<1 µM) by active transport and subsequent metabolism of GABA in neighboring neurons and glia. GABA catabolism in astroglia is rapid due in part to a highly active transaminase (Chan-Palay et al., 1979; Larsson and Schousboe, 1990) which maintains GABA at a low level in these cells. Thus under physiologically normal conditions a large concentration gradient exists between GABA in GABAergic neurons and the surrounding transporter-rich astroglia.

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6.2.1. In vivo Detection of GABA using NMR GABA is readily detected in vitro using 1 H and 13 C NMR following enrichment with 13 C-labeled isotopes (Behar et al., 1983; Bachelard, 1998; Westergaard et al., 1995; Patel et al., 2001; Badar-Goffer et al., 1992). In vivo detection of GABA is possible using modern NMR spectrometers although precise quantitation of GABA poses several challenges. Because the GABA concentration is low in brain, <1/10 the level of major amino acids such as glutamate or N -acetylaspartate, NMR detection of GABA in vivo has most often entailed detection of the more sensitive 1 H nucleus. The 1 H NMR spectrum of GABA (C4 H9 O2 N) consists of three methylene proton resonances (δ, ppm; 2.99t , 2.29t , 1.89q ) and all are multiplets (JHH ∼ 7 Hz), which exacerbates the extent of overlap with neighboring resonances. Macromolecules contribute to the 1 H spectrum of brain and interfere speciﬁcally with quantitation of GABA (Behar and Ogino, 1993); however, these resonances can be suppressed using T1 -nulling techniques (Behar et al., 1994). GABA can be quantiﬁed accurately in 1 H NMR spectra of animal and human brain in vivo by use of J -editing techniques (Rothman et al., 1984; 1993; Behar and Boehm, 1994; Preece et al., 1994; Preece and Cerdan, 1996; Shen et al., 2002; Kuzniecky et al., 2002; Hetherington et al., 1998; Terpstra et al., 2002). As generally applied, J -editing is used to select H4 and H2 resonances of GABA while suppressing other metabolites and macromolecules. These methods have been adapted to the quantiﬁcation of 13 C-labeled GABA and other metabolites in vivo using indirect detection of the 1 H nucleus (Fitzpatrick et al., 1990; de Graaf et al., 2003; see Chapter 3).

6.3. PATHWAYS OF BRAIN GABA SYNTHESIS AND CATABOLISM 6.3.1. GABA Metabolism in the GABA Shunt Glutamate is the major precursor of GABA in the mature mammalian brain (Roberts and Frankel, 1950; Noto et al., 1986). GABA synthesis from glutamate is catalyzed by the pyridoxal cofactor-dependent enzyme, glutamate decarboxylase (GAD) (Martin and Rimvall, 1993; Martin and Tobin, 2000), which is present in the cytoplasm of all known GABAergic neurons. GABA is oxidized to succinic acid in mitochondria by the sequential actions of GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) in a pathway known as the GABA shunt (Figure 6.2). The GABA shunt is quantitatively the major route of GABA synthesis and catabolism (Balazs et al., 1970). Because the glutamate precursors used in GABA synthesis are derived from α-ketoglutarate (αKG), a TCA cycle intermediate, this pathway provides an alternative route or ‘shunt’ for α-KG oxidation to succinic acid around two enzymes of the TCA cycle, α-ketoglutarate dehydrogenase and succinyl CoA synthetase. Oxidation of α-KG through the GABA shunt is less efﬁcient energetically than oxidation through the complete TCA cycle due to the loss of production of one molecule of GTP for each molecule of α-KG (glutamate) diverted through this pathway. The enzymes of the GABA shunt are expressed in both neurons and astroglia although the fraction of GABA catabolized through the shunt in each cell type is not clear. Thus, while GABA synthesis is conﬁned to neurons, GABA degradation can occur in both neurons and astroglia. As discussed below, results of 13 C-isotopic labeling studies suggest that astroglia play an important role in the catabolism of GABA and the supply of glutamate precursors for GABA synthesis in the form of glutamine. Enzymes of the GABA shunt also produce precursors for other neuroactive substances (Figure 6.3) including the psychoactive neurotransmitter/neuromodulator, gamma-hydroxybutyrate (Maitre, 1997) and GABA-conjugated peptides such as homocarnosine (Petroff et al., 2001). The combined ﬂux through these pathways, however, is small (less than a few percent) compared to the rate of GABA synthesis. These alternate pathways also appear to recycle GABA, in that GABA appears both as precursor and product, and thus are unlikely to be pathways of net GABA degradation.

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Ac-CoA

a-KG NAD

TCA Cy cle

Glu GAD

NADH 2

CO2

GTP GABA

GDP

Succinate

Succinyl-CoA

a-KG

GABA-T SSADH Succinic-SA NADH2

Glu

NAD

Figure 6.2. The GABA shunt pathway. The GABA shunt (shown in grey) bypasses two enzymatic steps of the TCA cycle resulting in the net loss of one molecule of GTP for each molecule of α-ketoglutarate metabolized to succinate through the shunt. GAD, glutamate decarboxylase; GABA-T, GABA-transaminase; SSADH, succinic semialdehyde dehydrogenase.

GABA

GABA-T

GABA-peptides SSR

SSADH NADH NAD+ Succinic acid SSA ? DHHA

NADPH NADP+

GHBD

g-Hydroxybutyrate (GHB)

?

Figure 6.3. Pathways of brain GABA catabolism. The major pathway of GABA catabolism occurs in the GABA shunt, the enzymes for which are present in astroglia and GABAergic neurons. In this pathway GABA undergoes transamination with α-ketoglutarate to produce succinic semialdehyde (SSA) and glutamate. SSA is oxidized to succinic acid which enters the TCA cycle. GABA also serves as precursor for GABA-conjugated peptides, e.g., homocarnosine. The GABA catabolite, succinic semialdehyde (SSA) is the precursor of γ -hydroxybutyrate (GHB). Note, however, that the alternative pathways of GABA catabolism do not result in the net loss of GABA from the main oxidative pathway of the GABA shunt. Abbreviations: GABA-T, GABA-transaminase; SSADH, succinic semialdehyde dehydrogenase; SSR, succinic semialdehyde reductase; GHBD, γ -hydroxybutyrate dehydrogenase; GHB, γ -hydroxybutyrate; DHHA, 4,5-dihydroxyhexanoic acid.

6.3.2. Glutamate Decarboxylase and GABA Synthesis GABA is synthesized by α-decarboxylation of glutamate via glutamate decarboxylase (GAD). GAD activity has an absolute requirement for pyridoxal-5 phosphate cofactor. The interconversion between the cofactorbound and free forms of GAD serves as a major means of regulating GAD activity (Martin et al., 1991b). GAD occurs in brain as two major isoforms of 65 and 67 kDa (Erlander and Tobin, 1991; Martin and Rimvall, 1993). In the rat cortex GAD65 is the predominate form (∼70 %). The two forms are distributed differently within GABAergic neurons: whereas GAD67 is found throughout the neuron, GAD65 is highly enriched in nerve terminals and is associated with synaptic vesicles (Kaufman et al., 1991; Martin and Rimvall, 1993). GAD67 exists mainly as cofactor-bound active enzyme, whereas only about half of GAD65 is cofactor bound (Kaufman et al., 1991; Martin et al., 1991a,b). The differing kinetic properties and

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cellular distributions of the two GAD isoforms suggest different functional roles for cellular and vesicular GABA (Soghomonian and Martin, 1998; Belhage et al., 1993). NMR studies are beginning to shed new light on the function of these two isoforms in vivo (Section 6.6).

6.3.3. Alternate Pathways of GABA Synthesis Alternative but quantitatively minor pathways of GABA synthesis from putrescine and other polyamines have been described (Seiler, 1980). Polyamine pathways appear to play a quantitatively minor role in GABA synthesis (∼1 %) in the mature brain (Noto et al., 1986). Polyamines, however, may have a signiﬁcant role in the developing brain and retina (Yamasaki et al., 1999; Zawia and Harry, 1993), or possibly in the adult hypothalamus and other neuroendocrine associated regions, where the putrescine and potential GABA precursor, agmatine (4-aminobutyrl guanidine), is concentrated in unusually high levels (Sastre et al., 1996; Gorbatyuk et al., 2001).

6.4. ROLE OF GABA AND GLUTAMINE TRANSPORTERS IN GABA METABOLISM 6.4.1. GABA Transporters The ﬂow of GABA between neurons and astroglia is critical to the operation of the GABA/glutamine cycle (Figure 6.1). GABA transport proteins in plasma membranes of neurons and astroglia serve to maintain GABA at low extracellular levels, limiting diffusion from sites of release, and effectively terminating the receptor-mediated action of GABA (Henn and Hamberger, 1971; Iversen and Kelly, 1975; Hertz et al., 1977; Ryan and Roskoski, 1977). GABA transporters are Na+ and Cl− dependent and the most highly expressed ones in brain, GAT1 and GAT3, have a stoichiometry of 2Na+ and 1Cl− co-transported with each molecule of GABA (Kenyan and Kanner, 1988; Kavanaugh et al., 1992). Therefore, GABA transport is electrogenic and requires ATP to maintain the Na+ ion gradient and driving force for GABA uptake. The stoichiometry of ion and substrate transport for GABA transport in astroglia is shown in Figure 6.4. The role of the neuronal (GAT1) and astroglial (GAT1 and GAT3) GABA transporters with regard to the removal of synaptic and extra-synaptic GABA is not entirely clear. GAT1, which is the most highly expressed GAT and believed to mediate the majority of GABA transport (Schousboe, 2000; 2003), is present in high densities on axon terminals of GABA neurons and astrocytes near GABAergic synapses (Minelli et al., 1995; Conti et al., 1998). Inhibition of glial GAT1 results in a signiﬁcant increase in extracellular GABA (Juhasz et al., 1997), while inhibitors of astroglial but not neuronal GABA transporters are also effective anticonvulsants (Gadea et al., 2001; Schousboe, 2000; White et al., 2002). These studies suggest that astroglial transporters play an important role in the uptake and regulation of extracellular GABA. Astrocytes often show positive immunostaining with anti-GABA antibodies when GABA levels are elevated by inhibitors of GABA-transaminase (K. Behar and M. Schwartz, unpublished observations) underscoring the importance of astroglia in the removal and catabolism of GABA.

6.4.2. Glutamine Transporters Although glutamine has been known to provide precursors for GABA synthesis for many years, very little was known about how glutamine actually entered the cells. Signiﬁcant progress has been made recently in the characterization of glutamine transporters in both neurons and glia. Both electrogenic and electroneutral glutamine transporters are expressed in brain cells and both types involve co-transport of sodium ions. Glutamine transport into neurons involve A-type transporters (SA1 and SA2) and are electrogenic (Gln:Na+ = 1:1). In contrast astroglia express mainly the N-type (SN1) transporter which is electroneutral

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Astroglia NHE H+

H

+

Ac-CoA SN Gln Na+

2Na+ Glu

Cl− GAT GABA

GABA

TCA Cycle a-KG SSA

Figure 6.4. GABA and ion transport during GABA/glutamine cycling in astroglia. The depicted scheme shows substrate and ion ﬂows during GABA/glutamine cycling in astroglia. GABA inﬂux through the GABA transporter (GAT) co-transports 2 Na+ and 1 Cl− into the cytoplasm. Glutamine (Gln) efﬂux through System-N transporters (SN) removes 1 Na+ while counter-transporting 1 H+ into the cytoplasm resulting in an inﬂux of 1Na+ : 1H+ : 1Cl− per GABA/glutamine transport cycle. Hypothetical removal of H+ by coupled Na+ /H+ exchange could maintain H+ homeostasis at the expense of additional sodium loading. Thus, the degree of stimulation of Na+ /K+ -ATPase by Na+ inﬂux during the GABA/glutamine transport cycle could depend on astroglial pH regulation.

(Gln:Na+ : H+ = 1:1:1) due to counter-transport of H+ (Chaudhry et al., 2002). GABAergic neurons express both A-type transporters, although SA2 is particularly enriched in some interneurons. High densities of astroglial SN1 transporters surround GABAergic and glutamatergic processes (Boulland et al., 2002).

6.5. NMR STUDIES OF GABAERGIC FUNCTION 6.5.1. Measurements of GABA Synthesis Pharmacologic treatments that inhibit glutamate decarboxylase and GABA synthesis reduce GABA release and GABA-mediated inhibition (Golan et al., 1996), whereas electrical stimulation increases both GABA synthesis and GABA release (Corradetti et al., 1983). Thus, the rate of GABA synthesis is considered to reﬂect the activity of GABAergic neurons and their rate of energy utilization. The current primary methods for estimation of the GABA synthesis rate involve either administering a pharmacologic inhibitor of GABA catabolism (e.g., GABA-transaminase inhibitor) and measuring the initial rise in GABA over a deﬁned time period (Loscher et al., 1989; Behar and Boehm, 1994; Preece et al., 1994) or measuring the rate of GABA turnover using isotopically 13 C labeled precursors (Manor et al., 1996; Mason et al., 2001; Patel et al., 2003). In general, absolute GABA synthesis rates determined with GABA-transaminase inhibitors are less than values determined by the 13 C isotopic turnover of GABA. For example, the rate of GABA synthesis based on the initial rise of GABA in 1 H NMR spectra following GABA-transaminase inhibition is 0.03–0.04 µmol/g/ min, compared to 0.10–0.15 µmol/g/ min using [1-13 C]glucose (Mason et al., 2001). It is likely that the lower rate of GABA synthesis found when using GABA-transaminase inhibition is related to incomplete inhibition of the catabolic pathway, although feedback inhibition of GAD by rising GABA levels cannot be entirely excluded.

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6.5.2. GABA/Glutamine Cycling Mature neurons do not possess the necessary anaplerotic enzymes (e.g., pyruvate carboxylase) needed for net synthesis of glutamate precursors used in GABA synthesis. These precursors are derived primarily from astroglial glutamine (Paulsen et al., 1988; Badar-Goffer et al., 1990; 1992; Peng et al., 1993; Battaglioli and Martin, 1991, 1996; Sonnewald et al., 1993; Cerdan et al., 1990; Patel et al., 2001). While the importance of glutamine in GABA synthesis is in little doubt, quantitative information on the ﬂux of this precursor pathway in vivo was lacking. A study of the role of glutamine in net GABA formation in the rat cortex was conducted using [2-13 C]glucose, which labels glutamine-C3 via the astroglia-speciﬁc pyruvate carboxylase pathway (Patel et al., 2001). GABA synthesized from [3-13 C]glutamine precursors will lead to readily detectable 13 C labeling of GABA-C3. In the experiment GABA degradation and 13 C-isotopic label recycling was blocked by use of an inhibitor of GABA-transaminase. This study showed that ∼85 % of total GABA synthesis could be attributed to glutamine precursors (Patel et al., 2001). Preliminary results of an NMR study comparing GABA/glutamine and glutamate/glutamine cycling ﬂuxes at different levels of activity suggests that GABA/glutamine cycling ﬂux accounts for ∼22 % of total (GABA + glutamate) neurotransmitter cycling (Patel et al., 2003). Thus, GABA/glutamine cycling constitutes a quantitatively signiﬁcant pathway of GABA neurotransmitter repletion.

6.6. NMR STUDIES OF GABA SYNTHESIS REGULATION IN VIVO Quantitative NMR studies distinguishing contributions of the GAD isoforms to GABA synthesis have been reported recently. Manor et al. (1996) reported that the GABA synthesis rate was reduced when rats were treated with the GABA-T inhibitor vigabatrin, which increase GABA levels by blocking catabolism. Since GABA synthesis was reduced at 24 h but not 6 h of GABA elevation, feedback inhibition of GAD by the elevated GABA levels was unlikely. Rimvall and Martin (1994) have shown that one of the two isoforms of GAD (GAD67 ) is sensitive to GABA concentration and decreases as GABA levels rise. A quantitative re-analysis of the data of Manor et al. (1996) using a detailed metabolic model that included GABA/glutamine cycling with quantitative immunoblotting of the GAD67 and GAD65 isoforms from that study found that the 67–70 % reduction in VGAD was associated with a selective decrease of 44 % in GAD67 (Mason et al., 2001). Total GAD activity was reduced by 13 %, which could be accounted for entirely by the reduction of GAD67 protein. These ﬁndings provide evidence that GAD67 mediates the majority of cortical GABA synthesis under anesthetized (nonactivated) conditions, even though this isoform constitutes only in ∼30 % of total GAD (see Section 6.4.2). The NMR ﬁndings are supported by studies of GAD67 and GAD65 knockout mice, which show that the majority of brain GABA is a product of GAD67 (Asada et al., 1996a,b).

6.7. CONCLUSIONS GABA is synthesized primarily from glutamate and degraded in the GABA shunt pathway. While GABA synthesis occurs nearly exclusively in GABAergic neurons, GABA catabolism occurs in both neurons and astroglia. In vitro and in vivo NMR ﬁndings support the role of astroglial glutamine in the synthesis of GABA. An in vivo NMR study of the anesthetized rat cortex using an astroglial-targeted isotope ([213 C]glucose) indicated as much as 85 % of glutamate precursors used for net GABA synthesis arose from glutamine. Under anesthetized conditions ∼1/5 of the total cycling of glutamate and GABA with glutamine is contributed by GABA. Studies of the contributions of the two GAD isoforms to GABA synthesis also show that the bulk of cortical GABA synthesis under anesthetized (nonactivated) conditions is mediated by GAD67 . The combination of sophisticated NMR pulse techniques to select and localize GABA resonances with increased sensitivity and resolution provided by the new generation of high-ﬁeld (>7 T) magnets and

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gradients now available can be expected to provide new insights into the regulation of GABA metabolism and the GABA/glutamine cycle.

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7 Neural Energy Consumption and the Representation of Mental Events Simon B. Laughlin Department of Zoology, University of Cambridge, Downing St, Cambridge CB2 3EJ, UK

David Attwell Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK

7.1 7.2 7.3 7.4

7.5 7.6 7.7 7.8 7.9 7.10 7.11 7.12

Introduction Constructing the Signalling Energy Budget Neuronal and Glial Resting Potentials The Metabolic Cost of Signalling with Glutamatergic Synapses 7.4.1 Neurotransmitter Recycling 7.4.2 Vesicle Release and Recycling 7.4.3 Post-synaptic Energy Consumption 7.4.4 The Total Energy Used by a Synapse and its Distribution Among Synaptic Mechanisms 7.4.5 Total Cost of Synaptic Signalling by a Single Neuron The cost of Propagating Action Potentials in Grey Matter The Cost of Signalling with One Neuron Scaling Consumption by the Mean Firing Rate The Distribution of Energy Consumption Between Signalling Mechanisms The Robustness of our Estimates The Distribution of Mitochondria Supports our Estimate The Contribution of Neuronal Signalling to Cerebral Metabolic Rate The Division of Cerebral Metabolic Rate Between Signalling and Maintenance

Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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Brain Energetics and Neuronal Activity 7.13 The Inﬂuence of Energy Usage on Neural Representations 7.14 Implications of the Energy Budget for Functional Imaging

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7.1. INTRODUCTION The non-invasive techniques of functional imaging monitor local changes in cerebral metabolism and blood ﬂow. Because these local changes are associated with variations in neural signal level, functional images capture distributions of neural activity that are associated with events and actions. The brain’s ability to convert events into actions resides in its ability to represent and process information in neural circuits. Thus the extent to which functional imaging reveals mental function depends upon the relationships between the metabolic and vascular signals used for imaging, and the signals in neural circuits used by the brain. To understand these relationships it is important to know the quantities of metabolic energy involved in neural signalling because these quantities determine the strength and distribution of metabolic signals, and necessitate the changes in blood ﬂow and oxygenation that produce vascular signals. Neural signalling consumes signiﬁcant amounts of energy, consumption increases with activity and this increased demand is catered for by changes in metabolic rate and blood ﬂow. The fast neural signals, action potentials and post-synaptic potentials, are generated by ions moving across membranes and, to maintain concentration gradients, these ions are restored by pumps, co-transporters and exchangers. These homeostatic mechanisms derive their energy, directly or indirectly, from the hydrolysis of ATP to ADP, and most of the brain’s ATP is regenerated from ADP by the aerobic glycolysis of glucose. Sokoloff’s application of his 6-deoxy glucose method demonstrated that the rate of glucose consumption changes locally within the brain, and increases with neural activity (Clarke and Sokoloff, 1999). How is energy usage distributed among the many mechanisms that participate in neural activity? In 1975 Creutzfeld used biophysical data, the heat produced by action potentials in nerves, to calculate the energy required to transmit action potentials within cortex (Creutzfeld, 1975). Because of an arithmetic error, Creutzfeld’s calculation indicated that action potentials only accounted for approximately 1 % of the cortical metabolic rate, leading him to suggest that synapses were primarily responsible for activitydependent energy consumption. This conclusion placed metabolic signals at the sites at which information is processed, as opposed to transmitted, and was widely accepted. Experimental evidence supports a strong involvement of synaptic transmission in energy consumption. The density of mitochondria correlates with the density of the excitatory (glutamatergic) synapses (Wong-Riley, 1989; Wong-Riley et al., 1998) and, when activity is progressively suppressed by anaesthesia, a decline in the turnover of the synaptic glutamate is accompanied by a fall in glucose consumption (Sibson et al., 1998). However, changing the level of neural activity affects a wide variety of neural mechanisms, all of which use metabolic energy to a greater or lesser extent. To resolve the distribution of energy usage among neural mechanisms we undertook a comprehensive assessment of the energy used for neural signalling in cortical grey matter. We constructed a ‘bottom-up’ energy budget that, like Creutzfeld’s original calculation, takes biophysical and biochemical data from the literature to calculate the energy used by known signalling mechanisms as a function of action potential frequency. By multiplying these values by the numbers of sites at which they operate we estimated both the total energy requirement for neural signalling in grey matter as a function of ﬁring rate, and the distribution of this usage among signalling mechanisms. The budget demonstrates that the ion ﬂuxes used for electrical signalling dominate energy consumption and, contrary to Creutzfeld (1975), action potentials are as demanding as synapses. Biochemical pathways for neurotransmitter recycling are of secondary importance and, although evidence is slim, the same appears to be true of second messenger signalling.

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The details of the budget, the sources of the published data and the methods used for evaluation and calculation are in Attwell and Laughlin (2001). Here we summarise our approach and our ﬁndings, draw attention to critical assumptions, identify unresolved issues, and review more recent studies. We also consider how metabolic demands inﬂuence both the structure of neural representations and the ability of functional imaging techniques to determine this structure. Before proceeding we emphasise that the values in our budget are estimates that must be continually questioned and revised. Creutzfeld unwittingly misled the ﬁeld for 25 years because his convenient conclusion that action potentials make negligible demands on cortical energy was widely accepted. The overall pattern of energy usage revealed by our budget appears to be correct because the numbers of ions and molecules involved in the different types of signalling mechanism differ by orders of magnitude, but the absolute values and percentages are estimates that must not be set in stone.

7.2. CONSTRUCTING THE SIGNALLING ENERGY BUDGET We chose rodent cortex because it provides the best set of physiological and anatomical data. Much of the biophysical work on synaptic mechanisms has been carried out in rat and Braitenberg and Sch¨uz (1998) provide unique morphometric data on the density, structure and connectivity of neurons in mouse cortex. We calculated the energy used by the average cortical neuron (taken to be a pyramidal cell) when it signals with an action potential and restricted our estimate to the dendrites, cell body, axon collaterals and synapses in grey matter. The energy consumed by myelinated axons in white matter was not considered. We calculated the energy used when a neuron’s glutamatergic output synapses are stimulated by an action potential, the energy used to conduct this action potential through grey matter to the output synapses, and the energy used to maintain the resting potential, between action potentials. These values give the energy used for neural signalling as a function of action potential rate and identify the contributions made by mechanisms involved in signalling (e.g. action potentials, neurotransmitter recycling, vesicle recycling, post-synaptic currents, calcium transients in spines). Multiplying the energy consumed per neuron by the density of neurons in grey matter gives a speciﬁc metabolic rate that is comparable to measured values.

7.3. NEURONAL AND GLIAL RESTING POTENTIALS We made two assumptions: the resting potential is determined by the conductances and reversal potentials for sodium and potassium ions and the electrogenic action of the Na/K pump, and the resting cell is sufﬁciently compact that the input conductance measured by an electrode inserted in the cell body equals the total sodium and potassium conductance of the cell. Based on these assumptions we used published measurements of resting potential and input resistance to calculate sodium and potassium currents, pumps rates and hence ATP consumption. We calculated that a neuron hydrolyses 3.42 × 108 ATPs per second to maintain its resting potential, and a glial cell hydrolyses 1.02 × 108 ATPs/s. With approximately as many glial cells as neurons in grey matter, the total resting consumption, neurons plus glia, is 4.44 × 108 ATPs/neuron/s. We have classiﬁed the energy used to maintain the resting potential as a signalling activity because the resting potential ﬁlls in the gaps between action potentials. The resting potential can equally well be classiﬁed as a maintenance process that keeps a cell alive (Ames, 2000).

7.4. THE METABOLIC COST OF SIGNALLING WITH GLUTAMATERGIC SYNAPSES 7.4.1. Neurotransmitter Recycling When an action potential reaches a synaptic bouton one synaptic vesicle of glutamate is released with a probability that depends on mean ﬁring rate. The 4000 glutamates released from the vesicle are mainly

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taken up by astrocytes. Uptake of one glutamate is driven by the co-transport of 3 Na+ ions and 1 H+ , and the counter-transport of 1 K+ ion. The proton is restored by Na+ /H+ exchange, giving a net entry of 4 Na+ ions per glutamate. These sodium ions (and the potassium ion) are restored to the extracellular space by the Na/K exchange pump with a stoichiometry of 3 Na+ ions per ATP, giving an energy cost for uptake into astrocytes of 1.33 ATP per glutamate. We allowed 1 ATP per glutamate for metabolic processing in astrocytes (e.g. conversion to glutamine). The passive diffusion of processed product to neurons and the conversion back to glutamate are costless. Glutamate pumping into vesicles is powered by the vesicular proton ATPase which pumps at least one proton per glutamate at a cost of 1/3 ATP per proton (assuming that it operates like the mitochondrial F-type ATPase). Adding the costs of astrocytic uptake, metabolic processing and vesicle replenishment gave a total recycling cost of 2.66 ATP/glutamate which, with 4000 glutamates, gave a neurotransmitter recycling cost of 11 000 ATPs per released vesicle.

7.4.2. Vesicle Release and Recycling Measurements made at calyx synapses in the mammalian auditory system show that an action potential triggers a pre-synaptic inﬂux of 1.2 × 104 Ca2+ ions per vesicle released (Helmchen et al., 1997). Extruding these ions with the 3 Na+ /Ca2+ exchanger requires 1.2 × 104 ATPs. Thus the pre-synaptic calcium inﬂux that stimulates vesicle release is about as costly as neurotransmitter recycling. On thermodynamic grounds the fusion of the vesicle to the membrane requires the equivalent of 10.5 ATPs (Siegel, 1993), giving an absolute requirement of 21 ATPs per cycle of fusion and endocytosis. The intricate molecular machinery used for exocytosis and endocytosis is more costly to operate. Endocytosis involves around 400 molecules per vesicle (clathrins, adaptor proteins, dynamins and hsc70s) and, if each of these molecules is phosphorylated once, the cost is 400 ATPs per vesicle. Making the generous assumption that the molecular machinery for exocytosis (triggered by calcium inﬂux) is equally costly, the estimated cost of one cycle of exo- and endocytosis is of the order of 103 ATPs per vesicle, just 5 % of the combined cost of transmitter recycling and pre-synaptic calcium entry.

7.4.3. Post-synaptic Energy Consumption Post-synaptic energy consumption dominates the cost of synaptic transmission. The glutamate released from a vesicle binds to three types of receptors on the post-synaptic membrane, the non-NMDA and NMDA receptor channels, and G-protein coupled receptors. From measurements of channel conductance and open time and the electrochemical potential for the relevant ions, Na+ and Ca2+ , we calculated the numbers of ions entering the post-synaptic neuron when a channel is opened by binding glutamate, and then multiplied by the number of activated channels. Experimentally based estimates of the numbers of non-NMDA receptor channels activated by a vesicle vary from 15 to 200, giving a wide range of values of sodium inﬂux, 8.7 × 104 to 1.87 × 106 per vesicle. We took a weighted mean, based on the acknowledged possibility that the upper value is an overestimate (Markram et al., 1997), and calculated an inﬂux of 2 × 105 Na+ ions/vesicle. 67 000 ATPs must be hydrolysed to pump these Na+ ions back. Similarly, we estimate that 1.8 × 105 Na+ ions and 10 000 Ca2+ ions enter through NMDA receptor channels and 70 000 ATPs are required to return these ions. The response triggered by G-protein coupled receptors, a rise in intracellular free Ca2+ concentration in the post-synaptic spine, involves approximately 70 IP3 molecules and 3000 Ca2+ ions (both the spines and the intracellular calcium concentration are tiny) and the restoration of IP3 and Ca2+ ions gave a cost of a little over 3000 ATPs.

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7.4.4. The Total Energy Used by a Synapse and its Distribution Among Synaptic Mechanisms The distribution of energy usage among synaptic mechanisms illustrates two important points. First, energy costs are roughly proportional to the numbers of ions and molecules used for signalling. Second, when a synapse is an ampliﬁer, post-synaptic costs dominate. Releasing the vesicle involves 12 000 Ca2+ ions, requiring 12 000 ATPs. Ejecting and retrieving the vesicle involves hundreds of molecules requiring in the order of 1000 ATPs, and recycling the 4000 glutamates released from the vesicle requires 11 000 ATPs. The post-synaptic channels opened by the pre-synaptic release of one vesicle of glutamate admit close to 400 000 ions and a total of 137 000 ATPs are needed to pump them back out. Add to this the remarkably economical second messenger response, and the total post-synaptic consumption is 140 000 ATPs. The total energy consumption associated with the release of single vesicle of glutamate is 164 000 ATPs, 85 % of which is devoted to the post-synaptic response.

7.4.5. Total Cost of Synaptic Signalling by a Single Neuron On average a mouse pyramidal cell makes 8000 output synapses. At the estimated mean ﬁring rate in rodent cortex of 4 Hz, the probability that a spike triggers the release of a vesicle at a bouton is 0.25. On this basis the neuron releases an average of 2000 vesicles of glutamate per action potential, giving an estimated total synaptic cost of 3.28 × 108 ATPs/neuron/action potential.

7.5. THE COST OF PROPAGATING ACTION POTENTIALS IN GREY MATTER Synaptic vesicle release is stimulated by the arrival of an action potential. This electrical pulse travelled along axon collaterals in grey matter and, to make more distant connections, passed along the myelinated axon in white matter to (largely unmyelinated) terminal branches in grey matter. For statistical purposes these terminal branches are treated as collaterals (Braitenberg and Sch¨utz, 1998). The action potential also propagated over the cell body and depolarised the dendrites. Taking data on the membrane areas of collaterals, cell body and dendrites we estimated the inward current required to charge the membrane capacitance to the peak of action potential (assumed to be a change of 100 mV). The total Na+ inﬂux is larger than the capacitive current because K+ ions are leaving the neuron as Na+ ions enter. We multiplied the capacitive Na+ inﬂux by 4, the factor for squid giant axon (Hodgkin, 1975). We converted this inﬂux to energy consumption by assuming that it requires 1 ATP to pump out 3 Na+ ions. Our analysis of pump kinetics showed that 3–10 % less ATP is required because the Na+ ﬂux is inﬂuenced by changes in concentration and membrane potential, but we did not apply this small correction. Transmission along axon collaterals accounts for 82 % of the energy used by action potentials. The average pyramidal cell makes 4 cm of axon collateral including terminal branches (Braitenberg and Sch¨utz, 1998) with mean diameter 0.3 µm, necessitating an inﬂux of 9.44 × 108 Na+ ions. Invading the 25 µm diameter spherical cell body requires 4.92 × 107 Na+ ions. In line with recorded responses and models of pyramidal cells we have assumed that dendrites are depolarised on average by 50 mV, consequently 1.57 × 108 Na+ ions are needed to depolarise the 4 mm of dendrite of mean diameter 0.9 µm. Adding these contributions (and neglecting the myelinated axon in white matter), a total of 1.15 × 109 Na+ ions enter a neuron per action potential, and pumping them out takes 3.84 × 108 ATPs. Any involvement of Ca2+ ions in dendrite depolarisation affects this total by <10 %. Thus, contrary to Creutzfeld’s (1975) estimate, action potentials use slightly more energy than the synapses in rodent cortex. Our method of calculating

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action potential costs was checked by A. Roth and M. Hausser, using pyramidal cell models (Vetter et al., 2001) that incorporate the effects of ionic conductances and cell morphology.

7.6. THE COST OF SIGNALLING WITH ONE NEURON Adding the cost of transmitting an action potential to the cost of activating output synapses, the total cost of signalling is 7.1. × 108 ATPs/neuron/action potential. This cost per spike is equivalent to maintaining the resting potential of the neuron and associated glia for 1.6 s. Because the discharge of an action potential and the consequent activation of output synapses are metabolically signiﬁcant events, neural energy consumption rises steeply in proportion to spike rate from a relatively low baseline. This approximately linear relationship between spike rate and metabolic rate is observed when electrical activity is elevated or depressed experimentally (Smith et al., 2002; Clarke and Sokoloff, 1999). By coupling spike rate to neurotransmitter release, our model also predicts the linear relationship between metabolic rate and the turnover of synaptic glutamate observed in rat cortex by NMR spectroscopy (Sibson et al., 1998). The stoichiometry of the relationship between glutamate turnover and metabolic rate is disputed (Choi et al., 2002) and we note that a well known effect, a change in the probability of vesicle release with action potential frequency, will change the relationships between spike rate, energy consumption and the rate of turnover of synaptic glutamate.

7.7. SCALING CONSUMPTION BY THE MEAN FIRING RATE Although the mean ﬁring rate of cortical neurons is a critical parameter, both for setting the average level of usage (often called the baseline) and for interpreting the signiﬁcance of fractional increases in local metabolic rate (Hyder et al., 2002; Raichle and Gusnard, 2002; Smith et al., 2002), there are relatively few measurements from unrestrained animals in natural surroundings. The mean ﬁring rate in rat grey matter varies from 1.6 to 16 Hz, depending on the unit, and the means of populations vary from 1.5 to 4 Hz. Because the experiments were conducted in impoverished laboratory conditions we took the higher rate, 4 Hz, to obtain a consumption by action potentials and synapses of 2.85 × 109 ATPs/neuron/s. Adding the consumption by resting potentials gave a total consumption by a neuron and its associated glia of 3.29 × 109 ATPs/s.

7.8. THE DISTRIBUTION OF ENERGY CONSUMPTION BETWEEN SIGNALLING MECHANISMS The relative distribution of energy usage among signalling mechanisms in rodent grey matter is plotted in Figure 7.1 for an action potential rate of 4 Hz. 81 % of signalling energy is used to restore the ions that generated fast electrical signals, 47 % to action potentials and 34 % to post-synaptic responses. The slower G-protein coupled post-synaptic responses use much less signalling energy. Only 0.7 % is required to support calcium signals in post-synaptic spines, but other second messenger responses have not been budgeted for. Neuronal and glial resting potentials come second, with 13 % of consumption, 10 % for neurons and 3 % for glia. Recycling the neurotransmitter, glutamate, takes just 3 % and this small percentage has been conﬁrmed in conscious human brain by NMR spectroscopy (Choi et al., 2002). Pre-synaptic calcium entry also requires 3 % and the mechanisms for vesicle recycling (exocytosis and endocytosis) consume less than 0.5 %.

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Estimated distribution of ATP consumption mean spike rate 4 Hz Action potential 47 % propagation

34 % Post-synaptic ion fluxes

10 % r.p. - neurons 3% r.p. - glia Glu recycling 3 %

Pre-synaptic calcium 3 %

Figure 7.1. The distribution of ATP consumption among signalling mechanisms, calculated for grey matter of rodent cortex at a mean spike rate of 4 Hz (after Attwell and Laughlin, 2001).

7.9. THE ROBUSTNESS OF OUR ESTIMATES The hierarchy of energy usage, electrical signalling >resting potentials> chemical signalling, is ﬁrmly grounded in the biophysics and biochemistry of neural information processing. Post-synaptic potentials and action potentials dominate energy usage (81 % total) because relatively large numbers of ions pass through channels to produce electrical signals that must be sufﬁciently large to process information in the presence of noise and to travel long distances, and sufﬁciently fast to provide adequate temporal resolution and reaction times. In this respect it is interesting that auditory areas of the mammalian brain have higher speciﬁc metabolic rates (Sokoloff et al., 1977). One expects the restoration of pre-synaptic calcium (3 %) and the recycling of neurotransmitter (3 %) to use an order of magnitude less energy than the generation of post-synaptic responses (37 %) because the chemical synapse is an ampliﬁer. Post-synaptic second messenger responses use almost an order of magnitude less energy (0.7 %) than pre-synaptic mechanisms because these second messenger signals are short range and relatively slow. On this basis we dispute Ames’ proposal that second messenger signalling uses a signiﬁcant fraction of neural energy but agree that little quantitative data is available (Ames, 2000). Although the hierarchy of energy usage is well founded, our numbers are imprecise. Action potential usage is ﬁrmly based on the charging of the membrane capacitance and on morphometric data on membrane area (Braitenberg and Sch¨utz, 1998). Moreover, the correct application of Creutzfeld’s (1975) method of calculating cortical energy usage from measurements of the heat produced by action potentials in peripheral nerve gives similar results (Attwell and Iadecola, 2002). We have identiﬁed two uncertainties. The ﬁrst is that the action potential propagates throughout the axonal arbour. Failure at branch points will reduce energy consumption. The second is the multiplication of the capacitive current by a factor of 4, to account for the shunting of inward sodium current by outward potassium current. We have assumed that this ratio applies to all excitable membrane in grey matter but the sodium and potassium conductances of the 0.3 µm diameter axonal collaterals are unknown quantities.

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Synaptic costs are subject to two uncertainties. The ﬁrst is the number of post-synaptic ion channels activated by the release of a vesicle of neurotransmitter which is reported to vary over a wide range and could, in principle, vary from synapse to synapse. Better measurements of a larger number of glutamatergic pyramidal cell synapses are required. The second uncertainty is the proportion of synapses that release transmitter in response to an action potential. The probability of release varies considerably, according to the previous history of signalling. Setting a higher release probability increases both the fraction of energy consumed by synapses and the consumption per neuron per action potential (Lennie, 2003). We have probably underestimated the energy required to maintain resting potentials. We employed resistance measurements made at or close to the cell body of an assumed isopotential neuron to estimate the resting Na+ inﬂux. However, because a proportion of the average pyramidal cell’s 4 cm of axonal arbour lies well beyond the limits of an electrically compact cell, this underestimates the energy required for neural resting potentials. A truer estimate requires data on the projection and membrane properties of axon collaterals. For glia we assumed that the lower input resistances reported in the literature result from electrical coupling and, if this is not the case, the glial contribution is underestimated. Thus estimates of resting consumption are likely to be revised upwards as better data become available. Indeed, the latest results from NMR spectroscopy suggest a more substantial contribution from glia, albeit in the deeply anaesthetised brain (Choi et al., 2002).

7.10. THE DISTRIBUTION OF MITOCHONDRIA SUPPORTS OUR ESTIMATE Mitochondria are located where ATP is used, and in grey matter they are concentrated around synapses, in dendrites and pre-synaptic terminals (Wong-Riley, 1989). These locations suggest that synapses use much more energy than action potentials. However, the large number of pre-synaptic mitochondria are in a position to satisfy two requirements, the modest needs of pre-synaptic mechanisms (<10 % of total) and the larger amount of energy (34 % of total) required to support action potential propagation in axon collaterals. Because the average distance between synaptic terminals along collaterals is 5 µm (Braitenberg and Sch¨utz, 1998), the diffusion of ATP and ions to and from the pre-synaptic terminal can provide for the axon (Attwell and Iadecola, 2002). The short length of axon and associated synapse could be a unit for energy consumption and supply in which, as in the insect photoreceptors (Stavenga and Tinbergen, 1983), the inﬂux of Ca2+ ions at the pre-synaptic terminal stimulates the mitochondrion in anticipation of the demands made by ion pumps. Given that the mitochondria in pre-synaptic terminals and collaterals provide for action potential transmission through the axonal arbour, the distribution of mitochondria in grey matter agrees well with our estimate of energy usage (Figure 7.2).

7.11. THE CONTRIBUTION OF NEURONAL SIGNALLING TO CEREBRAL METABOLIC RATE We converted the rate at which one neuron uses energy to signal to a speciﬁc metabolic rate (signalling usage per gram of neocortical grey matter) by multiplying by the neuronal density, 9.2 × 107 neurons/cm3 (Braitenberg and Sch¨utz, 1998) to give 30 µmol ATP/g/min. This calculation assumes that all neocortical neurons are pyramidal cells–a reasonable ﬁrst approximation because approximately 80 % of neurons are pyramidal cells, and 90 % are glutamatergic. The remaining 10 % of inhibitory neurons would have to have very different patterns of energy usage for this assumption to produce serious errors. Our estimate of 30 µmol ATP/g/min is close to the range of rates measured in different areas of rat grey matter, 33–50 µmol ATP/g/min. These rates were calculated from measurements of glucose utilization tabulated by Clarke and Sokoloff (1999) by assuming that, because of proton leak, only 31 ATPs are generated per glucose (Rolfe and Brown, 1997). The similarity between our estimates and measured usage (Figure 7.3)

Neural Energy Consumption and the Representation of Mental Events Predicted energy usage Observed mitochondria

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0 Dendrites

Axons + Synapses

Glia

Figure 7.2. A comparison between the distribution of energy usage and the distribution of mitochondria among dendrites, axons plus synapses, and glia in grey matter. Energy usage is calculated for rodent grey matter spiking at 4 Hz and the mitochondrial distribution is taken from data on monkey visual cortex (Wong-Riley, 1989). The ﬁgure is replotted from Attwell and Laughlin (2001). Specific ATP consumption for grey matter estimated as a function of average spike rate

Specific consumption µmoles ATP /g/min

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20 Signalling r.p. Non-signalling 0 0

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4 6 Average spike rate Hz

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Figure 7.3. The speciﬁc ATP consumption of rodent grey matter estimated as a function of spike rate. The maintenance consumption is based on measurements in which all electrical activity is blocked and is assumed to be independent of spike rate (see text for further details). The contributions of neuronal plus glial resting potentials (r.p.) and signalling are from the bottom up energy budget (Attwell and Laughlin, 2001). Signalling includes both synaptic transmission and the propagation of action potentials in grey matter.

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suggests that a large proportion of the energy used by grey matter goes to sustain signalling. How realistic is this suggestion?

7.12. THE DIVISION OF CEREBRAL METABOLIC RATE BETWEEN SIGNALLING AND MAINTENANCE The proportion of cerebral metabolic rate devoted to signalling has been estimated by blocking neural transmission with deep anaesthesia, and by blocking the Na/K pump which uses almost all the energy required to support signalling. These manipulations reduce the energy consumption of the whole brain, or large regions of the brain, by approximately 50 % (Astrup et al., 1981; Siesj¨o, 1978), suggesting that the energy consumption of the brain as a whole is equally divided between signalling and maintenance. This same result is obtained when NMR spectroscopy is applied to large volumes of rat brain (Choi et al., 2002) but when NMR measurements are conﬁned to cortex, blocking signalling depresses the metabolic rate by 80–90 % (Sibson et al., 1998). The discrepancy between NMR measurements can be explained by attributing the higher metabolic rate in rat grey matter (30–45 µmol ATP/g/min versus 20 µmol ATP/g/min for the brain as a whole) to signalling. If, like the whole brain, grey matter uses 10 µmol ATP/g/min for maintenance, the remaining 20–35 µmol ATP/g/min is used for signalling and this range of values is in good agreement the budget’s prediction of 30 µmol ATP/g/min. We suggest therefore that 75 % of grey matter’s metabolic rate is used for signalling (Figure 7.3). We cannot account for most of the 10 µmol ATP/g/min that we have attributed to maintenance. Of the many processes that contribute to the viability and structural integrity of neurons, glia and blood vessels, few have been measured in vivo. The brain has a relatively low rate of protein turnover, and from published measurements in rat we calculated that protein synthesis uses 0.4 µmol ATP/g/min. Ames (2000) produces a similar estimate for rabbit retina. Nucleotide turnover generally uses less energy than protein synthesis (Ames, 2000) and phospholipid metabolism could consume as much as 1 µmol ATP/g/min (Attwell and Laughlin, 2001). These estimates suggest that macromolecular synthesis accounts for approximately 20 % of the energy used for maintenance. Actin hydrolysis has been suggested to use 50 % of the energy consumed by neurons cultured from chick ciliary ganglia (Bernstein and Bamburg, 2003), but this percentage cannot be extrapolated to intact mature brain because the technique used to measure ATP consumption involved blocking ATP production, which is known to block synaptic and action potential signalling, i.e. the major energy consuming processes in grey matter. Thus, the Bernstein and Bamburg data suggest only that the cytoskeleton uses 50 % of energy after synaptic and action potential signalling are blocked, which is about 20 % of the total energy consumption. Nonetheless, cytoskeletal maintenance and intracellular transport are good candidates for consuming a substantial fraction of the maintenance energy, and should be examined further (Ames, 2000). We calculated that the energy needed to counteract the leakage of protons from synaptic vesicles is relatively small, <0.3 µmol ATP/g/min. Proton leakage from mitochondria should be given serious attention because in some tissues it accounts for 20 % of resting energy consumption (Rolfe and Brown, 1997).

7.13. THE INFLUENCE OF ENERGY USAGE ON NEURAL REPRESENTATIONS Rodent grey matter has a high speciﬁc metabolic rate, equal to that of active heart muscle (Ames, 2000) and human leg muscle running the marathon (Hochachka, 1994). The budget shows that most of this metabolic energy is used to transmit fast electrical signals relatively long distances along axon collaterals, and across large numbers of synapses. Thus the metabolic demands of grey matter are directly related to the ability of cerebral cortex to represent and associate large numbers of diverse events and actions. High metabolic

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demands can profoundly inﬂuence the way in which information is represented and processed (Levy and Baxter, 1996), by limiting the number of available action potentials (Lennie, 1998). Our calculations prove that the limit is severe. The metabolic rate of rodent grey matter supports a mean ﬁring rate of 4 Hz, and to support 18 Hz the metabolic rate equals the maximum sustainable rate of mammalian striated muscle (Attwell and Laughlin, 2001). Lennie applied our method of calculating energy usage to human brain (Lennie, 2003), and estimated that its metabolic rate can support a mean ﬁring rate of approximately 0.16 Hz. Lennie’s estimate for humans is lower than our estimate for rodent for three reasons. The human speciﬁc metabolic rate is lower than rat (as expected from the difference in body mass), Lennie’s calculations assume that half the metabolic rate of cortical grey matter goes to maintenance whereas we assumed 25 %, and the proportion of synapses activated per action potential is assumed to be 0.5 in primates, compared with 0.25 in rat. Lennie (2003) predicts that approximately 50 % of signalling energy is consumed by synapses as opposed to 33 % for spike propagation. We also predicted that the proportion of energy consumed by synapses is higher in primate cortex than in rodent cortex. We based our prediction on the larger number of synapses per neuron, and noted, but did not allow for, the extension of the axonal arbour and the longer dendrites needed to connect these synapses. Allowing for longer collaterals and dendrites will increase action potential costs, and return the percentage consumption by synapses to a value closer to the rodent’s. Lennie’s (2003) increase in synaptic usage is largely due to the two-fold higher probability of transmitter release at boutons. Given that metabolic costs limit signalling, brains may well have evolved to be metabolically efﬁcient. Neurons, circuits and codes should be adjusted to reduce the costs of signal transmission and synaptic transfer, and events and actions should be represented by as few action potentials and active synapses as possible. There is prima facie evidence that cortex increases metabolic efﬁciency by these means. Fine axon collaterals (average diameter 0.3 µm) reduce the number of ions required to transmit an action potential by reducing membrane area. The orderly arrangement of neurons in maps has a similar effect because it reduces the distance that action potentials must travel. Sparse distributed codes reduce the numbers of action potentials required to represent events and actions, and the optimum degree of sparseness depends upon the balance between the cost of signalling and ﬁxed costs of maintaining neurons ready to signal (Levy and Baxter, 1996; Attwell and Laughlin, 2001). For optimum efﬁciency <15 % of a population of cortical neurons should be activated to represent a new event or action in rodent and <4 % in human (Attwell and Laughlin, 2001; Lennie, 2003). Reducing the probability that synapses release transmitter can save large amounts of energy. In rat, if the probability of transmitter release increased from 0.25 to 1.0, the energy used per cortical neuron per action potential would more than double and the permissible ﬁring rate would halve. In principle, synaptic plasticity can reduce synaptic drive, and hence cost, without losing information because using 8000 synapses to drive the spike output of a single pyramidal cell is massively redundant (Levy and Baxter, 2002). A metabolically efﬁcient cortex should use synaptic plasticity to ensure that the signal delivered by every synapse adds information that is relevant to the post-synaptic neuron’s output. Cortical neurons exhibit other means of reducing redundancy, such as brief transient responses to sustained stimuli, adaptation to local signal level and subtraction of the local mean (Attwell and Laughlin, 2001). Neural mechanisms associated with attention also improve efﬁciency by restricting the volume of cortex in which activity is heightened (Lennie, 2003).

7.14. IMPLICATIONS OF THE ENERGY BUDGET FOR FUNCTIONAL IMAGING The ability of functional imaging techniques to monitor the processing of information in neural circuits depends upon their sensitivity and resolution. Because an action potential has a signiﬁcant impact on

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a neuron’s energy requirements and the baseline ﬁring rate is low, metabolic measures are sensitive to small changes in neural activity. We estimated that signalling at 4 Hz accounts for 75 % of metabolic rate; consequently when 1 % of neurons increase their rate from 4 Hz to 40 Hz the energy demand increases by 7.5 %. Like the BOLD signal (Logothetis et al., 2001), energy demand increases linearly with spike rate and the rate of activation of synapses but this does not necessarily mean that BOLD is a direct response to the rate at which signalling is consuming energy (Heeger and Ress, 2002). It appears that blood ﬂow is increasing in response to neural activity in anticipation of increased energy demands. Evidence is accumulating that neurotransmitter release controls cerebral blood ﬂow and these ﬁndings emphasise that the sensitivity and kinetics of the BOLD signal are determined by the cell signalling systems that connect neural activity to blood vessels (Zonta et al., 2003; Bonvento et al., 2002; Attwell and Iadecola, 2002). Our ﬁndings are consistent with the evidence that inhibition produces a local decrease in energy demand (Shmuel et al., 2002). Because most neurons are excitatory, the smaller number of inhibitory neurons are very effective at counterbalancing excitation. Consequently a small increase in the activity of inhibitory neurons can produce a larger decrease in the activity of excitatory neurons, leading to an overall reduction in energy consumption. If the brain has compensated for the high energy demands of neural signalling by using sparse distributed codes, the neural representation of events will involve relatively small numbers of action potentials that appear transiently in a small proportion of neurons. In distributed codes, events and actions are speciﬁed by the particular combinations of neurons that are active. Thus a number of different representations will generate identical metabolic signals because they activate the same proportion of neurons in a given population. BOLD signals will be particularly badly compromised by sparse distributed coding because spatial and temporal resolution are limited by the arrangement of blood vessels (Harrison et al., 2002) and the signalling mechanisms that link neural activity to vascular control (Attwell and Iadecola, 2002). Neural wiring patterns in the cortex can, in principle, limit the resolution of metabolic signals. Axon collaterals and synapses distribute approximately 80 % of a pyramidal cell’s signalling consumption locally within grey matter, over distances in the order of 1 mm (Braitenberg and Sch¨utz, 1998). Thus the neural signals that represent events and convert representation into action are not only sparse, but diffuse, and these properties make it difﬁcult to decipher how neural circuits process signals to generate neural representations of events and actions If one knows how neural circuits work, one can relate changing patterns of bloodﬂow and energy usage to the activity of speciﬁc neurons (Lauritzen, 2001). The critical role played by a knowledge of circuitry is not obvious in our published energy budget (Attwell and Laughlin, 2001) because we considered the average distribution of signals in grey matter. Nonetheless, we followed neural signals through a deﬁned network to determine the energy used for signalling. This network of average cortical neurons is closed (the number of intracortical connections greatly exceeds the number of ﬁbres entering and leaving the cortex) and, by considering the average ﬁring rate, we placed the network in a steady state. In a closed network at steady state the average number of synaptic inputs to a neuron equals the average number of its synaptic outputs and, assuming that spikes do not fail within neurons, input and output synapses are equally active. These properties of a closed steady-state system enabled us to deﬁne the pattern of energy usage without detailed descriptions of speciﬁc circuits. Detailed descriptions of speciﬁc circuits are essential for interpreting functional images, when these are images of local deviations from steady state. Under transient conditions there is no reason why the numbers of action potentials entering a voxel should equal the number leaving, and one cannot equate the number of activated input and output synapses. To infer patterns of neural activity from changes in local metabolic rate one must know which neurons are activated, how they are connected, how they drive each other and what mechanisms they use. It follows that the ability of functional imaging to reveal the neural basis of mental acts is limited by our understanding of the anatomy and physiology of neural circuits.

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Acknowledgements Supported by the Wellcome Trust, a Wolfson–Royal Society award, and the European Union (DA), and the Rank Prize Fund and the BBSRC (SBL).

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Rolfe, D. F. S. and Brown, G. C. (1997) Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiological Reviews, 77, 731–758. Shmuel, A., Yacoub, E., Pfeuffer, J., Van de Moortele, P.-F., Adriany, G., Hu, X. and Urgubil, K. (2002) Sustained negative BOLD, blood ﬂow and oxygen consumption response and its coupling to the positive response in the human brain. Neuron, 36, 1195–1210. Sibson, N. R., Dhankar, A., Mason, G. F., Rothman, D. L., Behar, K. L. and Shulman, R. G. (1998) Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proceedings of the National Academy of Sciences of the United States of America, 95, 316–321. Siegel, D. P. (1993) Energetics of intermediates in membrane fusion: comparisons of stalk and inverted micellar intermediate mechanisms. Biophysics Journal, 65, 2124–2140. Siesj¨o, B. (1978) Brain Energy Metabolism, Wiley, New York. Smith, A. J., Blumenfeld, H., Behar, K. L., Rothman, D. L. and Shulman, R. G. (2002) Cerebral energetics and spiking frequency: the neurophysiological basis of fMRI. Proceedings of the National Academy of Sciences of the United States of America, 99, 10765–10770. Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O. and Shinohara, M. (1977) The [14 C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure and normal values in the conscious and anesthetized albino rat. Journal of Neurochemistry, 28, 897–916. Stavenga, D. G. and Tinbergen, J. (1983) Light dependence of oxidative-metabolism in ﬂy compound eyes studied in vivo by microspectroﬂuorometry. Naturwissenschaften, 70, 618–620. Vetter, P., Roth, A. and H¨ausser, M. (2001) Action potential propagation in dendrites depends on dendritic morphology. Journal of Neurophysiology, 85, 926–937. Wong-Riley, M. T. T. (1989) Cytochrome-oxidase - an endogenous metabolic marker for neuronal- activity. Trends in Neurosciences, 12, 94–101. Wong-Riley, M. T. T., Anderson, B., Liebl, W. and Huang, Z. (1998) Neurochemical organization of the macaque striate cortex: correlation of cytochrome oxidase with Na+ K+ ATPase, NADPH-diaphorase, nitric oxide synthetase and N-methyl-D-aspartate receptor subunit I. Neuroscience, 83, 1025–1045. Zonta, M., Angulo, M. C., Gobbo, S., Rosengarten, B., Hossmann, K.-A., Pozzan, T. and Carmignoto, G. (2003) Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nature Neuroscience, 6, 43–50.

8 Imaging Cerebral Metabolic Rate of Oxygen Consumption (CMRO2) using 17 O NMR Approach at Ultrahigh Field Wei Chen, Xiao-Hong Zhu and Kamil Ugurbil Center for Magnetic Resonance Research, Department of Radiology, University of Minnesota Medical School, 2021 6th St. SE, Minneapolis, MN 55455, USA

8.1 Central Role of Oxidative Metabolism in Brain Function and Brain Diseases 8.2 Existing Methodologies for Determining CMRO2 in vivo 8.2.1 Positron Emission Tomography Approach 8.2.2 13 C NMR Approach 8.3 17 O NMR Approaches and CMRO2 Measurements 8.3.1 Direct and Indirect 17 O NMR Approaches for CMRO2 and CBF Measurements 8.4 Increased 17 O NMR Sensitivity at Ultrahigh Fields 8.4.1 17 O Relaxivity and Sensitivity as a Function of Field Strength 8.5 Advantages of Direct 17 O NMR Approach for Imaging CMRO2 8.6 Ultrahigh Field 17 O NMR Approach for Imaging CMRO2 8.6.1 Theoretical Aspects of 17 O NMR Approach for Imaging CMRO2 8.6.2 Experimental Determination of CMRO2 using 17 O NMR Approaches 8.7 Conclusions and Perspectives

Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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8.1. CENTRAL ROLE OF OXIDATIVE METABOLISM IN BRAIN FUNCTION AND BRAIN DISEASES In the brain, the majority of the energy consumption occurs by neuronal activity (Attwell and Laughlin, 2001). This energy need is met predominantly through oxygen consumption mediated by the mitochondrial respiratory chain (Siesjo, 1978) which ultimately catalyzes the chemical reaction: 4H+ + 4e− + O2 −−−→ 2H2 O

(8.1)

The energy available from this reaction is subsequently utilized to generate the high-energy phosphorous metabolite adenosine triphosphate (ATP). The oxidative energy need in the human brain to sustain neuronal activity constitutes a large fraction of the energy requirements in the entire body at basal conditions. Although the weight of the human brain is only ∼2 % of the body weight, ∼20 % of the body’s oxygen consumption occurs in the brain. The carbon source that supports this oxidative metabolism is predominantly glucose, which is oxidized according to the chemical reaction: Glucose + 6O2 −−−→ 6H2 O + 6CO2

(8.2)

A small amount of glucose can be metabolized nonoxidatively in the brain to form the lactate molecules (Figure 8.1). The oxidative process, however, provides at least 15 times more ATP than the nonoxidative pathway. Under resting conditions, the glucose metabolism is almost completely through oxidation (Siesjo, 1978, and references therein). Basal cerebral oxygen consumption is not uniform across different regions in the brain. It is recognized that capillary density in the brain is inhomogeneous (Harrison et al., 2002, and references therein), suggesting that mitochondrial density, and hence, oxygen consumption are likely to be inhomogeneous since a good correlation exists between capillarity and mitochondrial density in tissues (Hoppeler and Kayar, Glc CMRglc

CBF (CBV)

CMRO2

Non-oxidative pathway

Oxidative pathway

Metabolism

HbO2

Susceptibility H2O + CO2

Lactate

O2 + Hb

BOLD Hemodynamics & Oxygenation

Figure 8.1. Schematic relationships among metabolic, hemodynamic and oxygenation responses during physiological resting and activation conditions. The glucose (Glc) compound is transported from blood plasma into the brain tissues and is consumed through two main pathways: (i) the oxidative pathway leads to the ﬁnal products of CO2 and H2 O and (ii) the nonoxidative glycolysis pathway leads to the ﬁnal product of lactate. The cerebral metabolic rates of glucose and oxygen are represented by CMRglc and CMRO2 , respectively. Both CMRO2 and CBF (and/or CBV) changes can affect the oxygenation level, ultimately susceptibility, and lead to BOLD contrast during neuronal activity. Zhu et al. (2002c). Reproduced with permission from the National Academy of Sciences, U.S.A.

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1988). It is evident that there are tight correlations under basal conditions among different brain tissue compartments (e.g., gray matter versus white matter) and among different brain regions (Siesjo, 1978; Raichle, 1987) between the cerebral metabolic rates of oxygen and glucose consumption (CMRO2 and CMRglc , respectively) as well as between these two metabolic rates and cerebral blood ﬂow (CBF) (Siesjo, 1978; Raichle, 1987). Alteration in neuronal activity elevated by sensory stimulation and/or performance of a task further increases spatial nonuniformity due to regional changes potentially in all of these parameters (i.e. CMRO2 , CMRglc and CBF) (e.g., Fox et al., 1988; Fox and Raichle, 1986; Hyder et al., 1996; Chen et al., 2001; Raichle, 1987). Questions related to the oxygen consumption rate are encountered frequently in biomedical research when considering either normal tissue function or abnormalities induced by diseased states. Relevant to normal brain function, a question of particular interest is whether the alterations in CMRO2 , CMRglc and CBF induced by neuronal activity is quantitatively coupled (or matched) (Barinaga, 1997). This question is central to understanding underlying mechanisms operative in most modern neuroimaging techniques. It has been suggested that the coupling between these metabolic and hemodynamic changes during brain activation are much more complex than that under basal conditions. Based on PET measurements (Fox and Raichle, 1986; Fox et al., 1988; Ribeiro et al., 1993), increases in CMRO2 were reported to be considerably less than those of both CBF and CMRglc during visual and somatosensory stimulations, suggesting that additional glucose utilization invoked by neuronal activity might be used nonoxidatively. This suggestion was partially supported by observations of lactate increase in the human primary visual cortex (V1) during visual stimulation (Prichard et al., 1992; Sappey-Marinier et al., 1992; Frahm et al., 1996). However, the quantity of the increased lactate was inadequate to explain the mismatch between the CMRO2 and CBF alterations observed in the PET measurements (Fox and Raichle, 1986; Fox et al., 1988; Ribeiro et al., 1993; Prichard et al., 1992; Shulman et al., 2001b; Shulman et al., 2001a). In contrast, several studies, including some that are based on the PET methodology, have provided data indicating that elevated neuronal activity could lead to a signiﬁcant increase in CMRO2 in the awake human brain (e.g., Roland et al., 1987; Marrett et al., 1993; Chen et al., 2001; Vafaee et al., 1999; Davis et al., 1998; Hoge et al., 1999), as well as in the anesthetized rat brain (e.g., Hyder et al., 1996; Kim et al., 1999). The discrepancies in the literature regarding the relationship among CBF, CMRO2 and CMRglc changes in response to elevated neuronal activity may have originated from differences in the nature of the stimulation employed, the cortical region involved, and the species used. In addition, however, the differences may have also arisen because of deﬁciencies in the neuroimaging approach used to perform the measurements. Accurate determinations of CMRO2 and CBF changes during brain activation are also crucial for quantiﬁcation of the Blood Oxygen Level Dependent (BOLD) contrast (Ogawa and Lee, 1990; Ogawa et al., 1990) and understanding the mechanism underlying the functional MRI (fMRI) approach based on the BOLD contrast (Ogawa et al., 1992; Blamire et al., 1992; Bandettini et al., 1992; Kwong et al., 1992). It is generally accepted that changes in CBF and CMRO2 lead to an increase in focal blood oxygenation level, a decrease in the blood deoxyhemoglobin content and ultimately a decrease in the magnetic susceptibility gradients near the luminal boundaries of the blood vessels near the activated site as illustrated in Figure 8.1 (Ogawa et al., 1998; Ugurbil et al., 1999; Chen and Ogawa, 1999). In this case, a signal increase in T2 ∗ and/or T2 -weighted MR images deﬁned as a positive BOLD effect can be detected. This model qualitatively provides a popularly accepted mechanism for interpreting the BOLD response during brain activation (Ogawa and Lee, 1990; Ogawa et al., 1998). However, at a quantitative level, the understanding of the relationship between BOLD contrast and neuronal activity requires reliable determinations of CMRO2 and CBF changes in response to brain activation. With such reliable and independent measurements, it should be possible to interpret BOLD results of fMRI study not only in normal brain but also under diseased conditions. For example, it has been reported that the BOLD increase in the human visual cortex during visual stimulation was signiﬁcantly greater in schizophrenia patients (4.6 ± 1.5 %) than that in normal

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subjects (3.1 ± 1.3 %), presumably because of impaired oxidative capacity of neurons in schizophrenia patients (Renshaw et al., 1994). Similarly, Huntington patients associated with mitochondrial impairment also have a greater BOLD change in response to visual stimulation (Jenkins et al., 1993) as compared to normal subjects. Direct measurements of CMRO2 in these patients should explicitly test the hypothesis that the reduced oxidative metabolism in the patients is the underlying cause of these BOLD observations. The central role of oxygen consumption is also evident in pathologies associated with the brain. Perturbations in brain oxidative metabolism have been linked to many brain diseases such as Schizophrenia, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, mitochondrial dysfunction as well as aging problems (Maurer et al., 2001; Wong-Riley et al., 1997; Maurer et al., 2000; Frackowiak et al., 1988; Beal, 1992). One line of evidence linking these diseases and cerebral oxidative metabolism is the histopathological ﬁndings indicating that the activity of cytochrome oxidase, the key mitochondrial enzyme that catalyzes the reduction of oxygen to form water, is signiﬁcantly impaired (>50 %) in Schizophrenic (Maurer et al., 2001) and Alzheimer’s patients (Wong-Riley et al., 1997; Maurer et al., 2000) in comparison with control groups. In addition, the impairment in the cytochrome oxidase content in the diseased brain was also found to selectively affect different regions in the brain (Maurer et al., 2001; Wong-Riley et al., 1997; Maurer et al., 2000). Therefore, the ability to image CMRO2 in vivo quantitatively is essential for efforts aimed at investigating cerebral oxidative metabolism under normal and pathological conditions as well as between basal and activation states.

8.2. EXISTING METHODOLOGIES FOR DETERMINING CMRO2 IN VIVO Due to the importance of oxidative metabolism in brain function and diseases, several methods have been developed to determine CMRO2 in the living brain. The earliest approach of CMRO2 measurement is based on the Kety–Schmidt method which relies on measuring CBF together with arteriovenous differences of oxygen content (Kety and Schmidt, 1948). This approach is relatively simple but involves substantial surgical preparations. It is thought to be the most accurate one in the literature. However, it provides global CMRO2 information without spatial differentiations within the brain.

8.2.1. Positron Emission Tomography Approach Positron Emission Tomography (PET) has been established for imaging CMRO2 in humans for many years (Ter-Pogossian et al., 1970; Mintun et al., 1984; Lenzi et al., 1981). By introducing oxygen gas enriched with the isotope 15 O(15 O2 ) into the human body and monitoring the spatial distribution and accumulation rate of the labeled water (H2 15 O) metabolized from the inhaled 15 O2 , PET can determine regional CMRO2 , and provide a 3D CMRO2 image. However, 15 O PET is unable to distinguish the radioactive signals between the 15 O2 molecule bound to hemoglobin and the 15 O atom incorporated into H2 15 O. To overcome this drawback, a measurement of cerebral blood volume (CBV) using an inhalation of C15 O has to be performed in addition to a CBF measurement based on intravenous injection of H2 15 O in order to determine CMRO2 accurately (Mintun et al., 1984). An alternative that eliminates a separate CBV determination has been suggested; this approach relies instead on time-weighted integration of data to estimate the vascular volume correction (Ohta et al., 1992). However, the modeling used to extract this information relies on numerous assumptions. For labeled water production to attain a constant value dictated by CMRO2 , oxygen concentration in tissue has only to exceed the Km of cytochrome oxidase; this may be attained rapidly so that even after labeled water production reaches a constant rate, 15 O2 –hemoglobin contents in the vasculature can be changing. Thus, even if an integration time window exists where the counts reﬂect predominantly vascular volume, it is not clear the vascular volume estimated from that time window is applicable to the time window where data are collected on labeled water production. In addition,

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utilizing this approach in CMRO2 calculations requires good signal-to-noise ratio in data acquired with high temporal resolution. These requirements complicate CMRO2 calculation based on PET data.

8.2.2.

13

C NMR Approach

The 13 C NMR approach is based on monitoring the intermediate labels incorporated from intravenously infused [1-13 C] glucose and provides a useful tool for measuring CMRO2 and other physiological parameters (e.g., Rothman et al., 1992; Novotny et al., 1990; van Zijl and Rothman, 1995; Hyder et al., 1996; Gruetter et al., 1998; Chen et al., 1998; Chen et al., 2001; Gruetter et al., 2001; also see Chapters 2–4). The labeling kinetics for several intracellular metabolites, most importantly glutamate, can be unequivocally measured by direct and/or indirect 13 C NMR techniques. The CMRO2 values can be derived based on the close correlation between the tricarboxylic acid (TCA) cycle rate and CMRO2 as well as the established modeling (Hyder et al., 1996; Chen et al., 2001; Gruetter et al., 2001; Mason et al., 1992, 1995). The 13 C NMR approach has been applied to study the CMRO2 changes in the rat somatosensory cortex during forepaw electric stimulation (Hyder et al., 1996, 1997; Shulman et al., 1999) and in the human visual cortex during photic stimulation (Chen et al., 2001). Another unique feature of the 13 C NMR approach is its capability for studying the glutamate/glutamine neurotransmitter release and cycling and functional bioenergetics during brain stimulation (e.g., Shen et al., 1999; Sibson et al., 2001; Rothman et al., 1999; Ugurbil et al., 2001; Gruetter et al., 2001; also see the details in Chapters 3 and 4). In general, the 13 C NMR approach requires a signiﬁcantly long measurement time (60–120 min) due to relatively slow turnover rates of cerebral metabolites involved in 13 C NMR measurements. In addition, it is difﬁcult to achieve 3D CMRO2 imaging with relatively high resolution, especially in the human brain, within a short measurement period because of the relatively long repetition time required for NMR signal acquisitions and the low inherent NMR sensitivity of 13 C NMR.

8.3.

17

O NMR APPROACHES AND CMRO2 MEASUREMENTS

8.3.1. Direct and Indirect Measurements

17

O NMR Approaches for CMRO2 and CBF

In principle, NMR-detectable, magnetic isotope 17 O can be utilized to measure CMRO2 . The concept underlying this idea is similar with that used in the 15 O PET approach. Explorations for monitoring labeled H2 17 O as a tracer of oxygen utilization or tissue perfusion using NMR spectroscopy and/or imaging techniques have been initiated a decade ago (e.g., Mateescu et al., 1988, 1989; Hopkins and Barr, 1987; Kwong et al., 1991; Arai et al., 1990; Pekar et al., 1991; Fiat et al., 1992; Pekar et al., 1995; Ronen and Navon, 1994; Ronen et al., 1998; Reddy et al., 1995). These studies can be separated into two distinct groups dependent upon the types of the NMR approaches used. The ﬁrst group is based on detecting H2 17 O directly (Mateescu et al., 1988, 1989; Arai et al., 1990; Pekar et al., 1991, 1995; Fiat et al., 1992). Nonlocalized 17 O NMR was employed for most studies in this group to estimate CMRO2 in the whole brain of experimental animals as well as in the human occipital lobe (Fiat et al., 1993), following an inhalation of the oxygen gas enriched with the 17 O isotope (17 O2 ). Some of these studies attempted to obtain a coarse CMRO2 image (∼0.8 ml voxel size) in the cat brain with a relatively long measurement time (15 min) (Pekar et al., 1991; 1995). Most of these in vivo studies were conducted at relatively low ﬁelds compared to what is available currently. These studies demonstrated the dramatic limitations imposed by the low inherent sensitivity of 17 O NMR due to its low gyromagnetic ratio, which is only 14 % of that of the proton nucleus. This sensitivity limitation has hindered further developments of the direct 17 O NMR detection approach, leading to the exploration of the second group of techniques that attempt to circumvent the sensitivity limitation by using indirect 17 O detection through the protons coupled to 17 O.

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The indirect 17 O detection approach relies on the effect of 1 H– 17 O scalar coupling on the transverse relaxation time (T2 ) or apparent T2 ∗ of the 17 O-coupled water protons. The T2 of water protons are considerably shortened by the presence of the 17 O nucleus in the water molecule (Meiboom, 1961; Hopkins and Barr, 1987). It has also been shown that the water proton T2 of H2 17 O can be restored to that of H2 16 O when 17 O decoupling is applied; in this case, the difference between proton signals obtained with and without 17 O decoupling is related to the 17 O content of the water sample (Ronen and Navon, 1994; Ronen et al., 1998; Reddy et al., 1995). However, the indirect 17 O measurements present difﬁculties for quantitatively correlating the proton signal changes to the absolute concentrations of H2 17 O since the water proton T2 is sensitive to many physiologic parameters, such as pH, the proton chemical exchange rate, temperature and magnetic ﬁeld strength. In addition, the estimated fractional change in 1 H signal induced by variations in metabolic H2 17 O concentration in the brain during an inhalation of 17 O2 is small at the basal condition. This fact could signiﬁcantly reduce the dynamic range of the indirect 17 O NMR approaches for determining CMRO2 . These approaches have so far not been very successful in imaging CMRO2 quantitatively.

8.4. INCREASED 8.4.1.

17

17

O NMR SENSITIVITY AT ULTRAHIGH FIELDS

O Relaxivity and Sensitivity as a Function of Field Strength

For a spin nucleus, signal-to-noise ratio (SNR) in a given acquisition time depends on its longitudinal relaxation time (T1 ), T2 ∗ , and the magnetic ﬁeld strength (B0 ) according to the following relationship SNR ∝ B0 β (T2 ∗ /T1 )1/2

(8.3)

where, based on theoretical considerations, the constant β was suggested to be 7/4 (Hoult and Richards, 1976; Wen et al., 1994). 17 O is the only stable oxygen isotope having a magnetic moment that can be detected by the NMR approach. It has a spin quantum number (I = 5/2) greater than 1/2 and possesses an electric quadrupole moment. The electric quadrupole moment interacts with local electric ﬁeld gradients and the temporal ﬂuctuations in this interaction induced by molecular motion lead to T1 and T2 relaxation processes (Abragam, 1961). In the case of water molecule, for which the extreme narrowing limit (i.e., τc ω << 1, where τc is the rotational correlation time and ω is Lamar frequency in radians) is applicable except for the bound water, the values of T1 and T2 can be estimated by using the following equation: 2 2 1 ∼ 1 3 2I + 3 η2 πe Qq = τc (8.4) 1+ = T2 T1 40 I 2 (2I − 1) 3 h where the term of (πe2 Qq/ h) is a quadrupole coupling constant and equals to 6.7 MHz (or 4.21 × 107 rad/s) for the 17 O nucleus, and η is an asymmetry parameter (0 ≤ η ≤ 1) (Abragam, 1961). For 17 O spins, thus, 1 ∼ 1 η2 12 = 42.53 × 10 (8.5) 1+ τc = T2 T1 3 Because both η and τc are independent of the magnetic ﬁeld strength, Equation (8.5) reveals that 17 O T1 and T2 (or T2 ∗ ) should be ﬁeld independent. If the τc value of 8.5 × 10−12 s is used for bulk water at 298 K (Steinhoff et al., 1993), then estimated T2 and T1 according to Equation (8.5) should be between 2.8 ms (for η = 0) and 2.1 ms (for η = 1). This estimation dictates that the 17 O relaxation times of water are short and in the range of several milliseconds. However, the actual 17 O T2 value should be smaller than the 17 O T1 value due to the signiﬁcant contributions of the 17 O– 1 H scalar coupling and the proton chemical exchange to the 17 O T2 (or T2 ∗ ) relaxation processes (Meiboom, 1961).

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We have experimentally and systematically investigated the longitudinal and transverse relaxation times as well as the signal-to-noise ratio (SNR) of 17 O spins at two ﬁeld strengths in both rat brain and saline solution using natural abundance water signals and 17 O MRS imaging based on direct detection (Zhu et al., 2001a). The relaxation times were found to be ﬁeld independent for the rat brain (T2 = 3.03 ± 0.08 ms, T2 ∗ = 1.79 ± 0.04 ms and T1 = 4.47 ± 0.14 ms at 4.7 T; and T2 = 3.03 ± 0.09 ms, T2 ∗ = 1.80 ± 0.06 ms and T1 = 4.84 ± 0.18 ms at 9.4 T), as well as for the saline solution at the room temperature (T2 = 4.28 ± 0.08 ms, T2 ∗ = 2.23 ± 0.04 ms and T1 = 6.59 ± 0.10 ms at 4.7 T; and T2 = 4.09 ± 0.06 ms, T2 ∗ = 2.19 ± 0.01 ms and T1 = 6.52 ± 0.12 ms at 9.4 T) (Zhu et al., 2001a). These results are consistent with the theoretical prediction that the dominant relaxation mechanism of 17 O nucleus is the quadrupolar interaction and is ﬁeld independent. The relaxation times measured experimentally are qualitatively in good agreement with the estimated values according to Equation (8.5). The ﬁeld independence of 17 O relaxivity is a major advantage for 17 O NMR at ultrahigh ﬁelds. Unlike proton spins in vivo, which are characterized by longer T1 and shorter T2 (or T2 ∗ ) with increased ﬁeld strength ultimately resulting in a reduction of sensitivity gain according to Equation (8.3), the ﬁeld independence of 17 O relaxivity means that 17 O sensitivity gain at higher ﬁelds is not compromised. Although the short T2 ∗ (i.e. the large linewidth) of the 17 O resonance leads to an effective reduction in the NMR sensitivity at any magnetic ﬁeld, this is compensated by the extremely short T1 value of the 17 O spins in H2 17 O, especially in the brain (< 5 ms) allowing rapid signal averaging. Therefore, it is possible to achieve a large sensitivity gain of 17 O signal at higher ﬁelds. The 17 O NMR sensitivity was evaluated at 9.4 T and 4.7 T in the rat brain and a saline solution (Zhu et al., 2001a). Figure 8.2 shows the sensitivity comparisons obtained by a surface coil in the rat brain at the two ﬁeld strengths. Figure 8.2(A) displays the localized signal proﬁle along one dimension parallel to (A)

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SNR9.4 T

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Figure 8.2. (A) One-dimensional SNR proﬁles of 17 O MRS imaging along an axis parallel to the RF coil plane in the rat brain at 4.7 T (top) and 9.4 T (middle), and the SNR ratio between the 9.4 and 4.7 T measurements. The average SNR gain at 9.4 T is approximately 4-fold in comparison with that at 4.7 T. (B) The 17 O NMR spectrum of natural abundance water from the central image voxel acquired from 4.7 T and 9.4 T, respectively, from the rat brain.

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(B)

Figure 8.3 (Plate 1). (A) Three-dimensional 17 O brain images of natural abundance H2 17 O from three adjacent slices (left color images) and corresponding anatomical images (right gray images) in the coronal orientation acquired from a representative rat. (B) Chemical shift image of natural abundance H2 17 O from the middle image plane as shown in (A). Zhu et al. (2002c). Reproduced with permission of the National Academy of Sciences, U.S.A.

the surface coil plane. Localized 17 O spectra acquired at 9.4 T and 4.7 T from a multidimensional chemical shift image (CSI) dataset corresponding to the same location in the rat brain relative to the surface coil are illustrated in Figure 8.2(B) (Zhu et al., 2001a). The striking ﬁnding from this study is the consistent SNR gain of approximately fourfold at 9.4 T as compared to 4.7 T indicating an approximate 7/4th power dependence of SNR on B0 . These experimental results demonstrate the signiﬁcant advantage provided by high ﬁeld strength for direct detection of 17 O NMR signal. This trend for increasing 17 O NMR sensitivity is expected to hold beyond 9.4 T. With the dramatic sensitivity gains realized at 9.4 T, it is possible to obtain a 3D 17 O CSI dataset (11 s of data acquisition and ∼0.1 ml voxel size) from the natural abundance water in the rat brain with good SNR despite the relatively low gyromagnetic ratio of 17 O nucleus (Zhu et al., 2002c). Figure 8.3(A) shows representative data obtained from three adjacent coronal slices in a rat brain showing anatomical images acquired by a larger 1 H RF coil and the 3D 17 O signal images of the natural abundance H2 17 O acquired by an 17 O RF coil and the Fourier series window imaging technique (Hendrich et al., 1994). Figure 8.3(B) displays one 17 O chemical shift image from the middle image slice as shown in Figure 8.3(A). The spatial distributions of 17 O signal intensity are not uniform because of the inhomogeneous B1 ﬁeld of the 17 O surface coil. Nevertheless, excellent SNR is evident with a relatively short acquisition time at 9.4 T, especially for the central voxels where SNR was optimized by the B1 proﬁle of the 17 O coil.

8.5. ADVANTAGES OF DIRECT FOR IMAGING CMRO2

17

O NMR APPROACH

There are several major advantages to using direct 17 O spectroscopic imaging to detect the H2 17 O distribution and its dynamic changes in the brain tissue. The short T1 of H2 17 O yields SNR gains per unit acquisition time as discussed previously. In addition, there is only a single, well-deﬁned H2 17 O resonance peak, consequently, making data analysis and quantiﬁcation simple and reliable. The H2 17 O signal from the natural abundance (0.037 %) water, which can be accurately imaged, provides an excellent internal reference for calculating absolute H2 17 O concentration or the H2 17 O concentration change following a H2 17 O injection or inhalation of 17 O2 in each CSI voxel. Such a simple quantiﬁcation is crucial for determining absolute CMRO2 values.

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The 17 O resonance of H2 O is relatively insensitive to B0 inhomogeneities (hence shimming performance) because of the intrinsically broad linewidth (>140 Hz) and the low gyromagnetic ratio. This implies that, compared to 1 H imaging, the requirements for B0 homogeneity either in the bare magnet, or together with room temperature shim compensation is considerably less stringent. The 17 O NMR approach for imaging CMRO2 is based on measuring the spatial distribution of the dynamic changes of the water metabolized from inhaled 17 O2 . Therefore, experimentally, the reliability and reproducibility of 17 O NMR in consecutively acquired datasets are far more crucial than the absolute sensitivity of the 17 O signal acquired in a single image or spectrum. In order to demonstrate this concept, we have conducted one study for comparing the signal ﬂuctuations in repeated measurements using direct and indirect 17 O NMR approaches. In this study, a 17 O– 1 H multinuclear surface coil probe was used to collect a series of global 17 O and 1 H spectra from the natural abundance water in the rat brain in order to estimate the relative signal ﬂuctuations (Zhu et al., 2002c). Two stacked plots of 25 17 O and 1 H spectra acquired consecutively using the 17 O coil and the 1 H coil, respectively, are shown in Figure 8.4. Although the number of acquisitions (NT) for each spectrum was different for the 17 O and 1 H nuclei, the repetition time (TR) was ∼3T1 in both cases and the total acquisition time for acquiring each spectrum was the same (6.2 s). Under this condition, we found that the signal ﬂuctuations in the consecutively acquired spectra for the two nuclei were comparable (SD = 0.20 % for 1 H spectra; SD = 0.35 % for 17 O spectra) even though the signal-to-noise ratio of the proton spectra from water was considerably higher. This is because the signal ﬂuctuations in the 1 H data are not dominated by the inherent sensitivity of a single spectrum or image but are mainly determined by the physiological ‘noise’ caused by breathing, heart pulsation, vasomotion, and spontaneous electrical and neurotransmitter activity, as well as NMR scanner instability. The similarity in signal ﬂuctuations in consecutively acquired 1 H and 17 O NMR spectra (Figure 8.4) indicated that, with respect to the capability for detecting dynamic changes in NMR signal intensity, the 17 O NMR method is comparable to the 1 H NMR method. Furthermore, these results suggested that direct 17 O detection might be more sensitive for measuring the changes in H2 17 O signal caused by the cerebral oxygen consumption of inhaled 17 O2 . This notion was based on the fact that a 17 O2 inhalation for 2 min under basal conditions in the anesthetized rat brain was expected to result in only 0.15–0.30 % change in the water proton signal intensity detected by the indirect 17 O detection approaches (Ronen et al., 1998; Zhu et al., 2002c), which is (A)

(B)

Figure 8.4. (A) Stacked plot of 25 1 H NMR spectra of water in the rat brain acquired and processed with following parameter: NT = 1, TR = 6.2 s, LB = 25 Hz (∼ half linewidth). The standard deviation of the 1 H water peak height in these spectra is 0.20 %. (B) Stacked plot of 25 17 O NMR spectra of natural abundance water in the same rat brain acquired and processed with the following parameters: NT = 512, TR = 12 ms for each scan, LB = 100 Hz (∼ half linewidth), and the standard deviation of the 17 O water peak height in these spectra is 0.35 %. The total acquisition times for each 1 H and 17 O spectra were the same (6.2 s).

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comparable to signal ﬂuctuations in consecutively acquired 1 H spectra or images. In contrast, the expected 17 O signal change for the same 17 O2 inhalation detected by the direct 17 O NMR approach is 20–40 %. These considerations provide the major justiﬁcation for pursuing the direct 17 O NMR approach at high magnetic ﬁelds for CMRO2 imaging. Both PET and 17 O NMR approaches for imaging CMRO2 are based on a similar concept of detecting the labeled water formed by oxidative metabolism of labeled O2 gas. However, the 17 O NMR approach overcomes one of the major difﬁculties associated with PET, namely the problem of distinguishing different forms of labeled oxygen simultaneously present in the tissue as O2 molecules bound to hemoglobin, as O2 molecules dissolved in tissue water and lipids, and as metabolically generated water. As discussed previously, in order to differentiate the radioactivity contributions from these different chemical species and precisely calculate CMRO2 , either two other PET measurements of CBF and cerebral blood volume are necessary or assumptions must be made about extracting this information from the temporal behavior of the label after a bolus inhalation. In contrast, 17 O NMR speciﬁcally detects the metabolically generated H2 17 O without confounding signals from the 17 O2 molecules bound to hemoglobin or dissolved in tissue space. When bound to hemoglobin, the 17 O2 resonance is broadened beyond detection due to the extremely slow rotational motion of the large molecular weight oxyhemoglobin complex in comparison with water. Saturation transfer electron paramagnetic resonance studies have shown that the τc value for rotational motion of the hemoglobin molecule is 2 × 10−8 s in solution and increases to 8 × 10−6 s when the hemoglobin molecule is encapsulated within the erythrocyte membrane (Cassoly, 1982). This is approximately 106 times slower than the rotational correlation time of water. Such slow rotational motion leads to extremely fast T2 relaxation according to Equation (8.5) and renders the 17 O2 molecule bound to hemoglobin invisible in 17 O NMR data. 17 O2 molecule in gas phase or dissolved in water is strongly paramagnetic due to its two unpaired electrons, and hence again undetectable because of the strong dipolar coupling between the electrons and the nucleus. 17 O signal becomes detectable only when 17 O2 is incorporated into water. Because of this chemical speciﬁcity, calculation of CMRO2 from 17 O NMR data is considerably less complicated as compared to the PET methodology (Mintun et al., 1984). In addition, unlike PET, the 17 O isotope is stable and nonradioactive. These advantages proclaim the feasibility of direct 17 O NMR approach at ultrahigh ﬁelds for mapping the spatial distribution of metabolic H2 17 O dynamically, and potentially for imaging CMRO2 noninvasively.

8.6. ULTRAHIGH FIELD

17

8.6.1. Theoretical Aspects of

O NMR APPROACH FOR IMAGING CMRO2 17

O NMR Approach for Imaging CMRO2

17

The dynamic change in cerebral H2 O concentration during an inhalation of 17 O2 is determined by three parallel processes: (i) oxygen consumption generating the metabolic H2 17 O in the brain, (ii) perfusion resulting in H2 17 O washout from the brain, and (iii) ﬂow recirculation bringing extra H2 17 O into the brain. These processes are illustrated in Figure 8.5. The mass balance equation of labeled H2 17 O concentrations during an inhalation of 17 O2 is given by dCb (t) = 2af1 CMRO2 + CBF{f2 [Ca (t) − Cv (t)]} dt

(8.6)

where Ca (t), Cb (t) and Cv (t) are the time-dependent H2 17 O concentrations in excess of the natural abundance H2 17 O concentration level in the arterial blood, brain tissue and venous blood, respectively; α is the 17 O enrichment fraction of inhaled 17 O2 gas. Proper use of Equation (8.6) requires consistencies of units among all terms used in this equation. The determinations of Cb , Ca and Cv are based on the 17 O NMR measurements and they can be calibrated using the natural abundance H2 17 O concentration (20.35 µmol

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135

Brain tissue CBF Artery

Vein Cb(t)

Ca(t)

Cv(t) CMRO2

Figure 8.5. Schematic diagram showing the mass balance of accumulated cerebral H2 17 O content (Cb (t)) contributed by oxidative metabolism of inhalated 17 O2 (CMRO2 ), ﬂow recirculation (Ca (t)) through feeding arteries and perfusion washout (Cv (t)) through veins according to Fick’s equation as described by Equation (8.6).

per gram brain water for brain tissue, and µmol per gram blood water for blood, calculated from natural abundance H2 17 O enrichment of 0.037 % and the molecular weight of H2 17 O = 19.0). Therefore, the preferred units are µmol/(g brain water) for Cb (t), and µmol/(g blood water) for Ca (t) and Cv (t). The unit used most commonly for CMRO2 is µmol/min/(g brain tissue) and is retained. This dictates the use of two unit conversion factors, f1 = 1.266 and f2 = 1.05, to achieve consistency of units among all parameters used in Equation (8.6) (Pekar et al., 1995; Zhu et al., 2002c). If water in the brain tissue is in equilibrium with water in the venous blood (i.e. fast water exchange across capillaries and membranes), f2 Cv (t) = Cb (t)/λ where λ is the brain–blood partition coefﬁcient (≈ 0.90) in units of (ml blood)/(g brain tissue) (Herscovitch and Raichle, 1985). Substituting this relation and introducing two new correction parameters (n and m) into Equation (8.6) leads to dCb (t) nCb (t) = 2af1 CMRO2 + mCBF f2 Ca (t) − (8.7) dt λ The solution of Equation (8.7) is t 2aλf1 mnCBF mnCBF CMRO2 1 − exp − t + f2 mCBF (t − t ) dt Ca (t ) exp − Cb (t) = mnCBF λ λ 0 (8.8) where m is a correction factor accounting for the limited permeability of water because water is not totally freely diffusible across the brain–blood barrier, and the value of m is 0.84 for the similar CBF range that we studied (Herscovitch et al., 1987). The constant n is another correction factor that accounts for another type of restriction on the permeability speciﬁcally for the H2 17 O generated through oxidative metabolism in the mitochondria (‘metabolic’ H2 17 O) during an inhalation of 17 O2 . This additional restriction is included in our modeling because we have observed that the washout rate of the metabolic H2 17 O after the cessation of 17 O2 inhalation was signiﬁcantly slower than the washout rate of the H2 17 O that permeates brain tissue subsequent to a bolus injection of H2 17 O through the internal carotid artery in the rat (Zhu et al., 2002a). The cause of this difference could be the permeability restrictions imposed by the inner and outer mitochondrial membranes that must be traversed by all water molecules generated by oxygen consumption. However, mitochondrial volume is a small fraction of the tissue volume; therefore, it is a small and likely undetectable perturbation for the washin and washout kinetics of labeled water that comes into the tissue space from the vasculature by exchange across the capillaries. Even when this exogenous labeled water equilibrates across all compartments in the tissue, extra-mitochondrial volume will contain most of that water and its washout will not be affected by the mitochondrial membranes. Irrespective of the

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validity of this explanation, however, the correction factor n that is used in modeling can be experimentally determined as the ratio of the washout rate of the metabolic H2 17 O after the cessation of 17 O2 inhalation versus the washout rate of the H2 17 O introduced after a bolus injection through the internal carotid artery (n < 1) (Zhu et al., 2002a). The CMRO2 value can be precisely calculated using the complete model as described by Equation (8.8) if all parameters of Cb (t), CBF, n and Ca (t) can be determined experimentally for the same animal brain. The ﬁrst three parameters can be determined using the 17 O NMR approaches. In principle, the Ca (t) function is determined by the total metabolic H2 17 O generated in the entire animal body. Ca (t) can be approximated as a linear function of the inhalation time (i.e., Ca (t) ≈ At, where A is a constant and t represents time) based on two assumptions: (i) the metabolic H2 17 O molecules generated in tissue space exchanges relatively rapidly with the blood compartment so that it is at or near equilibrium with the blood water1 , and (ii) the oxygen consumption rate is constant (i.e., time independent) in all tissues during a short 17 O2 inhalation time. This approximation was supported by our experimental results, which will be discussed later, and the literature. Then the solution of Equation (8.8) for calculating CMRO2 is −mnCBF −mnCBF Af2 λ2 mnCBF Cb (t) − t exp t + exp t −1 Af2 λt mn2 CBF λ λ λ − −mnCBF n t 1 − exp λ CMRO2 = 2aλf1 mnCBF

(8.9)

Therefore, according to Equation (8.9), the CMRO2 value at each data point measured at different inhalation times can be calculated from experimental data of CBF, A, n, Cb (t) and other known constants (f1 , f2 , m, α and λ) for each voxel. In general, the ﬁnal CMRO2 value is based on the average of all CMRO2 values except the ﬁrst two time points, that have been omitted because of their relatively large measurement errors and the fact that they may represent initial transient values as the 17 O2 concentration in the tissue space increases to a value above the Km of cytochrome oxidase. The ability to obtain CMRO2 measurements as a function of inhalation time and the use of only the time-independent CMRO2 values avoids this complication. A fundamental limitation of spatial resolution using the 17 O imaging approach will be water diffusion in the brain since water is usually assumed freely diffusible. The apparent diffusion constant (D) of water in the rat brain has been measured to be ∼10−3 mm2 /s (Lee et al., 1999) with small direction-dependent variations due to anisotropy. For a period of 2 min, which corresponds to the 17 O2 inhalation time we used, this translates to an average diffusion distance of 0.35 mm. However, the more relevant parameter is the diffusion distance during the ‘lifetime’ of an H2 17 O molecule in the brain tissue after it is formed and before it is washed out by blood ﬂow. This time is given by λ/CBF. Typically, CBF ∼ 1 ml/g/ min which means λ/CBF ∼ 1 min. Thus, we are concerned with diffusion distances of ∼0.25 mm. This is much smaller than the spatial extent of brain compartments and/or the brain regions activated by most brain stimulations. In addition, the metabolic water clearly is not freely diffusible in the brain tissue, possibly because of 1 When oxygen consumption rate is constant, the blood content of metabolically generated labeled water can increase linearly in time under many different kinetic scenarios only one of which is the condition of rapid exchange between the tissue and the blood compartment listed here. We have focused on this condition here only because it is generally accepted to be the case. In any case, direct experimental determinations we have performed have demonstrated that indeed the blood content of metabolically generated labeled water increases linearly in time.

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permeability barriers posed by the inner and outer mitochondrial membranes (Zhu et al., 2002a; Ohta et al., 1992). This could further reduce the diffusion distances within the same measurement time. During brain activation, CBF increases thereby further shortening the average ‘lifetime’ of the water molecule in tissue. Therefore, the diffusion of H2 17 O in the brain tissue is unlikely to be the major factor limiting spatial resolution of 17 O NMR imaging for determining CMRO2 and/or CBF. The major limitation on spatial resolution for imaging CMRO2 is still determined by the 17 O NMR sensitivity and the allowed inhalation time of 17 O2 . These same diffusion considerations also apply to PET measurements of CMRO2 with 15 O inhalation or even PET functional imaging using H2 15 O injection.

8.6.2. Experimental Determination of CMRO2 using

17

O NMR Approaches

The complete modeling (Equation (8.9)) for accurate calculation of CMRO2 requires several key parameters, which can be experimentally measured by using different methods combined with 17 O NMR. We have recently investigated the feasibility for developing the 17 O NMR approach for imaging CMRO2 in a small animal model during a brief inhalation of 17 O2 gas at 9.4 T. We have applied the 3D 17 O MRS imaging method to determine CBF after a bolus injection of H2 17 O through an internal carotid artery and the accumulation rate of H2 17 O content (Cb (t)) generated by oxidative metabolism in the rat brain during a 17 O2 inhalation for 2 min (Zhu et al., 2002c). The Cb (t) images were obtained, together with continuous experimental monitoring of the arterial input function (Ca (t)) by using an implanted carotid artery RF coil. The parameters measured in these experiments were used to calculate CMRO2 values in each voxel of the image using complete modeling that accounts for all contributions to the cerebral H2 17 O accumulated during an inhalation of 17 O2 . This study demonstrates the feasibility for imaging CMRO2 in the rat brain anesthetized with α-chloralose (Zhu et al., 2002c). The details for imaging CMRO2 we have employed (Zhu et al., 2002c) are the following: 8.6.2.1. CBF measurements CBF values were determined independently based on the ‘washout’ rate of the H2 17 O tracer in the brain tissue, following a rapid bolus injection of 17 O-labeled H2 17 O via one feeding carotid artery (Zhu et al., 2001a; Zhu et al., 2002c) according to Cb (t) = Cb (0) exp[−t (mCBF/λ)] + B

(8.10)

where Cb (0) is the signal intensity after the ﬁrst arrival of all labeled water following the bolus injection, and B is a constant. Fitting the exponential decay of the H2 17 O washout curve to Equation (8.10) gives the CBF value. CBF measurements can be performed by MR methods based on arterial spin tagging. However, the determination of the washout rate of exogenous (as opposed to metabolically generated) H2 17 O tracer in order to calculate the correction factor n requires the experiment performed with bolus injection of H2 17 O. The spatial localization of 17 O NMR was achieved by using the 3D Fourier series window technique (Hendrich et al., 1994). In this imaging method, the k-space sampling is weighted according to the Fourier coefﬁcients of a predetermined voxel shape to achieve an optimal ﬁlter applied at the acquisition stage. The short 17 O T1 value (4.5 ms) of cerebral H2 17 O in the rat allows rapid signal acquisition, and ultimately, more signal averages within a given sampling time (Zhu et al., 2001a). Therefore, a short TR of 12 ms (∼ 3T1 ) was used for gaining SNR. A short gradient echo time (TE = 0.4 ms) can be achieved by using a short phase-encoding gradient length. This short TE reduces the signal loss caused by the short 17 O T2 ∗ value (= 1.8 ms) in the rat brain at 9.4 T (Zhu et al., 2001a). The total acquisition time for acquiring each 3D image dataset was 11 s.

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The same 3D 17 O approach and acquisition parameters were used for imaging CBF and Cb (t) resulting in robust image registrations between the CBF and Cb (t) (or CMRO2 ) images with the same voxel size and position. For CBF measurements, one external carotid artery was catheterized for gaining access to the internal carotid artery without interrupting the blood circulation through the brain (Zhu et al., 2001a). A small quantity of enriched H2 17 O was rapidly injected into the brain via the internal carotid artery for CBF measurements using 17 O MRS imaging (Zhu et al., 2001a). Fitting the exponential decay of the H2 17 O trace in each image voxel according to Equation (8.10) determined the CBF value, which can then be used to generate 3D CBF images. 8.6.2.2. Cb (t) measurements during an 17 O2 inhalation It is necessary to collect 3D 17 O MRS images of the natural abundance 17 O labeled water before 17 O2 inhalation as a reference for quantifying the cerebral H2 17 O concentration changes during and after the 17 O2 inhalation. Figure 8.6 demonstrates stacked plots of 17 O spectra of cerebral H2 17 O from one representative voxel before (natural abundance), during and after an inhalation for 2 min of 17 O2 (72 % 17 O enrichment) gas (Zhu et al., 2002c). The 17 O signal intensity of cerebral H2 17 O is characterized by three distinct phases: (i) constant before the 17 O2 inhalation, (ii) approximately linear increase during the 17 O2 inhalation, and (iii) approximately exponential decrease after the cessation of 17 O2 inhalation and approaching a new steady concentration within a short recovery time (<10 min). The 17 O NMR sensitivity achieved at 9.4 T makes it possible to distinguish the different phases and to quantify Cb (t). 8.6.2.3. Ca (t) measurements during an 17 O2 inhalation The most challenging measurement faced by the complete model for calculating CMRO2 as described by Equation (8.9) is the experimental determination of Ca (t), the concentration of H2 17 O in the feeding artery, especially when a small animal model is used. One of the conventional approaches for determining Ca (t) is to continuously draw arterial blood samples (e.g., Pekar et al., 1995), where the 17 O tracer concentrations in the blood samples can be analyzed independently by using in vitro NMR, mass spectroscopy or other methods. This approach is technically limited in studies of small experimental animals due to several factors: (i) only a small quantity of blood volume in the animal body available for blood draws, (ii) difﬁculty in manually drawing blood samples inside a small-bore animal magnet, (iii) the low accuracy in determining the blood sampling time and the absolute tracer concentration, thus resulting in large variations in Ca (t) measurements, and (iv) possible changes of physiological condition caused by blood removal (e.g., decrease in blood pressure). To overcome these limitations, we have designed an implanted 17 O RF coil that permits F2 (cm) −0.4 −0.2 −0.0 0.2 0.4 0.6 0.8

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Figure 8.6. Stacked plots of the cerebral H2 17 O spectra from one representative voxel as indicated by the cycle in the anatomical image (left insert) acquired before (natural abundance), during (as indicated by the dark bar under the stacked plots) and after 17 O2 inhalation for 2 min. Zhu et al. (2002c). Reproduced with permission of the National Academy of Sciences, U.S.A.

Imaging Cerebral Metabolic Rate of Oxygen Consumption (A)

(B)

139

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Figure 8.7. (A) 17 O spectrum of natural abundance H2 17 O in the rat carotid artery blood (∼7 µl) obtained using the implanted RF coil before inhalation of 17 O2 . (B) Time course of 17 O MR signals of Ca (t) in one carotid artery (ﬁlled circle) and Cb (t) from a representative voxel in the rat brain (open square) during inhalation of 17 O2 .

the continuous measurement of Ca (t) in a rat carotid artery (Zhang et al., 2003). This coil is based on a modiﬁed solenoid coil design combined with an RF shield. The RF shielding ensures that the NMR signal detected by the implanted coil originates only from the arterial blood (∼7 µl) without contaminations from surrounding tissues. Therefore, additional spatial localization is not necessary for determining Ca (t). In addition, the RF shielding minimized the electromagnetic coupling between the implanted 17 O coil and the head 17 O surface coil tuned at the same operating frequency (two-coil conﬁguration); this allowed simultaneous measurements of both Ca (t) and Cb (t) using two independent receiver channels with the same temporal resolution (11 s). Figure 8.7(A) illustrates the natural abundance H2 17 O spectrum detected from the blood in the rat carotid artery. Figure 8.7(B) plots the results of simultaneous measurements of Ca (t) change in one carotid artery and Cb (t) change in a voxel located in the brain from the same rat during an inhalation of 17 O2 . The results indeed demonstrate a linear relation between Ca (t) and the inhalation time t. The linear regression of Ca (t) data gives the value of the constant A used in Equation (8.9) for each 17 O2 inhalation measurement. 8.6.2.4. Calculation of CMRO2 and generation of 3D CMRO2 images Finally, the values of Cb (t), CBF and n from each voxel and the value of A measured from each 17 O inhalation measurement in the same subject can be used to calculate CMRO2 according to Equation (8.9). Figure 8.8 demonstrates one example of CMRO2 calculation as a function of inhalation time from a single voxel in the rat brain. It is evident that the CMRO2 value is independent of the inhalation time if the initial two points are excluded. The typical behavior of CMRO2 versus the inhalation time as demonstrated in Figure 8.8 reveals that the calculated CMRO2 reaches a constant value during early inhalation times indicating a rapid approach of the tissue concentration of 17 O-labeled oxygen molecule to Km of cytochrome oxidase. On the other hand, this experimental observation also validates the accuracy of the complete model for calculating CMRO2 . Information similar to that shown in Figure 8.8 can be obtained for all voxels in the 17 O image and then can be used to generate 3D CMRO2 images in the rat brain. Figure 8.9 illustrates three adjacent CMRO2 images in the coronal orientation from a representative rat brain. The averaged CMRO2 and CBF values in the rat brain from seven measurements (ﬁve rats) were 2.19 ± 0.14 µmol/g/ min and 0.53 ± 0.07 ml/g/ min, respectively, under conditions of α-chloralose anesthesia (Zhu et al., 2002c).

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CMRO2 (µmol/g/min)

4 3 2 1 0 −1 0

0.5

1 1.5 Time (min)

2

Figure 8.8. Plot of the calculated CMRO2 values using the complete modeling as described by Equation (8.9) as a function of inhalation time.

Figure 8.9 (Plate 2). Three-dimensional coronal CMRO2 images of rat brain measured by using 17 O NMR approach during 17 O2 inhalation for 2 min.

Due to the technical challenges faced by most existing CMRO2 imaging methods, there is virtually no literature reporting direct measurements of 3D CMRO2 image in the rat brain. Nevertheless, we compared our result of CMRO2 measurements using the 17 O NMR approach with the literature results obtained by indirect methods under similar animal condition. Based on an autoradiographic study (Nakao et al., 2001), cerebral metabolic rate of glucose (CMRglc ) was estimated to be ∼0.37 µmol/g/ min in the somatosensory and motor cortices of rats anesthetized with α-chloralose. Using the CMRO2 /CMRglc ratio of 5.5–6.0 (Siesjo, 1978), CMRO2 values of 2.04–2.22 µmol/g/ min are calculated from this CMRglc value. Our CMRO2 result (2.19 ± 0.14 µmol/g/ min) is perfectly within this range. The CBF value of 0.58–0.68 ml/g/min reported in the same autoradiographic study (Nakao et al., 2001) is also close to the mean CBF value of

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0.53 ± 0.07 ml/g/ min measured in our study. The 13 C heteronuclear editing MRS and 1 H MRI methods have been used to measure CMRO2 and CBF values in the sensory motor cortex of mature rats covering a large range of cortical activity under different anesthesia conditions, and a linear correlation between CMRO2 and CBF was observed (CMRO2 ≈ 3.76 × CBF + 0.18, R 2 = 0.99) (Hyder et al., 2000). The estimated CMRO2 from this linear relation is 2.16 µmol/g/ min for the mean CBF value observed in our study (0.53 ml/g/min). This estimated CMRO2 value is again in agreement with our result. These comparisons provide mutual support between our direct CMRO2 measurements and other indirect measurements reported in the literature, and conﬁrm the validity of the high-ﬁeld 17 O NMR approach for 3D CMRO2 imaging in small animal brains. The increased 17 O NMR sensitivity at 9.4 T has signiﬁcantly improved the spatial resolution (∼0.1 ml voxel size) for 3D CMRO2 imaging in the rat brain with a much shorter inhalation time (2 min) compared with the previous study conducted at 4.7 T (Pekar et al., 1995) which reported single voxel (0.8 ml voxel size) CMRO2 results based on 2D 17 O CSI in the cat brain with 15 min of inhalation time (Pekar et al., 1995).

8.7. CONCLUSIONS AND PERSPECTIVES The superior 17 O NMR sensitivity achieved at ultrahigh ﬁelds makes it possible for detecting and imaging small dynamic changes of metabolic H2 17 O during a brief inhalation of 17 O2 gas, and ultimately, for imaging CMRO2 in three dimensions in the brain. All key parameters involved in the complete model for calculating CMRO2 can be determined by independent and concurrent 17 O NMR measurements in the same animal. The CMRO2 results obtained by the 17 O NMR approach are consistent with the literature results for similar measurement conditions. The promising in vivo work conducted at ultrahigh ﬁelds provides a crucial step toward the goal for developing a robust and noninvasive 17 O NMR approach for imaging CMRO2 in animal, and potentially in human brains. Meanwhile, the work also provides rich information about other interesting aspects of 17 O NMR for CMRO2 imaging. As demonstrated in Figure 8.6, the cerebral H2 17 O concentration reaches a new steady state within a short time (6–10 min) after the cessation of 17 O2 inhalation. This fast recovery allows repeated CMRO2 measurements in the same subject and experimental session (Zhu et al., 2001b). This capability is essential for studying the oxidative metabolism changes related to perturbations in physiology and function, where at least two measurements are required under control and perturbation (or stimulation) conditions. Such a capability has been demonstrated in a preliminary study conducted at 9.4 T showing a focal CMRO2 increase in the rat somatosensory areas during the forepaw electric stimulation (Zhu et al., 2002b). This preliminary result indicates a great potential of high-ﬁeld 17 O NMR approach for studying the central role of oxidative metabolism in brain function, as well as for understanding the mechanisms underlying fMRI (Barinaga, 1997; Ogawa et al., 1998). Furthermore, the same approach can be applied for numerous applications for addressing different physiological and pathological questions. We have recently tested this notion through the investigation of the feasibility of 3D 17 O MRS imaging at 9.4 T for determining CMRO2 changes in the rat brain at normal and low brain temperature. The results indicate a signiﬁcant decrease of CMRO2 across the entire brain when the brain temperature dropped several degrees from the normal brain temperature. The capability of 17 O MRS imaging at ultrahigh ﬁelds for multiple measurements of 3D CMRO2 imaging is promising for measurements of CMRO2 changes elevated by perturbation of physiological condition. It should be interesting to apply the same 17 O NMR approach to study other perturbations, such as hypoxia and ischemia, as well as brain diseases, and potentially to study other living organs beyond brain. Another interesting ﬁnding related to Figure 8.8 is the potential of 17 O NMR for achieving a much faster temporal resolution for CMRO2 calculation according to Equation (8.9) using a single time point

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measurement (not valid for the initial points due to large calculation errors when the inhalation time is too short) if the ﬂuctuation of the measured CMRO2 value without averaging is tolerable. The conﬁrmation of this potential could signiﬁcantly strengthen the capability of 17 O NMR approach for achieving fastest in vivo 3D imaging of CMRO2 , and ultimately for studying the temporal changes of CMRO2 during physiological perturbations and/or brain stimulations. There is evidence showing that the increase of CMRO2 during a sustained stimulation might vary as a function of stimulation time (Mintun et al., 2002). The high-ﬁeld 17 O NMR approach with high temporal resolution should provide a useful tool for examining this aspect. The CMRO2 measurements described in this chapter involve two invasive procedures, one for determining Ca (t) by means of the implanted 17 O RF coil, and the second for measuring CBF by an intra-arterial catheter and bolus injection of H2 17 O. The CBF measurement can be performed noninvasively using the arterial spin tagging MR approaches (Detre et al., 1994; Kim, 1995; Kwong et al., 1995). The use of the implanted RF coil, however, is not suitable for routine measurements of CMRO2 using small experimental animal models (e.g., rat or mouse), or for human applications. Therefore, it is important to further explore the feasibility of 17 O NMR approach for reliably imaging CMRO2 without resorting to invasive Ca (t) and/or CBF measurements. Recently, we have investigated and showed this feasibility for imaging CMRO2 in the rat brain using simpliﬁed models, which only require noninvasive measurements of the cerebral H2 17 O concentration (i.e., Cb (t)), showing similar CMRO2 results to those obtained using the complete model accounting for all parameters (Zhang et al., 2002). This work opens the possibility to establish a completely noninvasive 17 O NMR approach for CMRO2 imaging. However, this possibility needs further validations in different physiological conditions and species. This is, in particular, interesting and important for potentially human applications, where elimination of invasive procedures is desirable if not absolutely necessary. The success of high-ﬁeld 17 O NMR for imaging CMRO2 in the human brain again relies on the 17 O NMR sensitivity. The sensitivity loss caused by the use of large RF coil size could be partially compensated by increasing image voxel size in the humans, which have a much large brain size in comparison with most animals. Recently, we have studied the in vivo NMR sensitivity for acquiring 3D 17 O images of natural abundance H2 17 O in the human visual cortex at 7 T with a temporal resolution of 8.5 s and a voxel size of 6.6 ml. Figure 8.10(A) demonstrates the results obtained from a representative subject showing one coronal 17 O CSI of natural abundance H2 17 O (extracted from 3D 17 O CSI dataset). The CSI plane (A)

(B)

2000

−1000

Hz

SNR ≈ 60

Figure 8.10. (A) 17 O CSI of natural abundance H2 17 O in the coronal orientation acquired from the human visual cortex at 7 T. (B) One representative 17 O spectrum from one central voxel as shown in (A) with 8.5 s of image acquisition time and 6.6 ml voxel size.

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was located in the human visual cortex. Figure 8.10(B) displays a representative 17 O spectrum from one central voxel showing the resonance peak of natural abundance H2 17 O in the human visual cortex with excellent SNR similar with that obtained from the rat brains at 9.4 T albeit with a smaller absolute voxel size but not relative to the size of brain (Zhu et al., 2001a, 2002c). Furthermore, the dynamic concentration change of metabolic H2 17 O during an 17 O2 inhalation is expected to be similar between the awake human brain and the rat brain anesthetized with α-chloralose because of the similar values for both CMRO2 (2.2 µmol/g/ min in the anesthetized rat brain versus 1.7 µmol/g/ min in the awake human visual cortex) and CBF (0.53 ml/g/min in the anesthetized rat brain versus 0.54 ml/g/min in the awake human visual cortex) (Zhu et al., 2002c; Fox et al., 1988). This comparison indicates that conditions in the rat brain we studied and the awake human brain are similar relative to the parameters that impact determination of CMRO2 from the 17 O data, and it will be possible to achieve 3D imaging of CMRO2 in the human brain at 7 T or higher magnetic ﬁelds. Realization and utilization of this potential could have an important impact on studying bioenergetics in the human brain. The CMRO2 imaging using the 17 O approach is expected to be more speciﬁc (i.e., accurate) for functional mapping of brain activation with respect to delineation of the actual sites of evoked neuronal activity. The major reason is that a CMRO2 image can reﬂect the metabolic change induced by brain activation and this change is tightly coupled with the neuronal activity (Siesjo, 1978; Raichle, 1987). In principle, the 17 O approach can circumvent many of the concerns about the speciﬁcity of currently used fMRI techniques, which are confounded by multiple contributions from different physiological changes during brain activation, and it can evolve as one of the most accurate functional imaging modalities. However, the spatial resolution of 3D CMRO2 image achieved currently is still far lower than that of BOLD-based and CBF-based fMRI as well as the CMRglc imaging based on autoradiographic approach (e.g., (Nakao et al., 2001)). The 17 O sensitivity gain both at 9.4 T for animal applications and at 7 T for human applications is still inadequate for functional mapping of CMRO2 changes at the spatial level of sub-cortical organizations such as the iso-orientation columns in the cat brain and the ocular dominance columns in the human brain (Bonhoeffer and Grinvald, 1993; Bonhoeffer and Grinvald, 1991; Kim et al., 2000; Horton and Hedley-White, 1984; Menon et al., 1997; Goodyear and Menon, 2001; Buchert et al., 2002; Cheng et al., 2001). It should be feasible to apply the high-ﬁeld 17 O approach for mapping and assessing CMRO2 differences between distinct brain compartments (e.g., gray matter versus white matter) as well as between activated and control brain regions especially at the spatial resolution available from the 17 O NMR approach. Furthermore, there is room for gaining more 17 O NMR sensitivity, consequently, for going to higher spatial resolution of 3D CMRO2 imaging by improving RF coil efﬁcacy and NMR acquisition strategies as well as NMR methodology, by averaging multiple measurements (a common strategy used for most neuroimaging modalities) and by further increasing ﬁeld strength. In conclusion, the ultrahigh-ﬁeld NMR systems currently available or in development for both animals and humans provide exciting opportunities for MR applications in medicine, especially for those nuclei with low gyromagnetic ratio. One of these nuclei that beneﬁt the most from ultrahigh ﬁeld strength is 17 O NMR, which shows great potential for in vivo imaging CMRO2 noninvasively in both animals and humans.

Acknowledgments We are grateful for the great assistance and support from Hao Lei, Nanyin Zhang, Yi Zhang, Run-Xia Tian, Xiaoliang Zhang, Hellmut Merkle, Jae-Hwan Kwag, Haiying Liu, Soeng-Gi Kim, Itamar Ronen and John Strupp. We also wish to acknowledge Gheorghe D. Mateescu, Gil Navon, Robert G. Shulman, Douglas L. Rothman, Seiji Ogawa, Joseph J.H. Ackerman and Alan C. McLaughlin for their encouragements and scientiﬁc discussions. This work was supported by NIH RO1 grants of NS41262, NS38070, NS39043, EB00329, EB00513 and P41 RR08079 (a National Research Resource grant from NIH), MIND Institute and the W.M. Keck Foundation.

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9 Deriving Changes in CMRO2 from Calibrated fMRI Fahmeed Hyder Departments of Diagnostic Radiology and Biomedical Engineering, Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06510, USA

9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 9.10

Introduction Physiology of Neuroimaging Neuroimaging with MRI Biophysics of fMRI Methods for Calibrating fMRI Physiological Basis of fMRI Neural Basis of fMRI Implications for Human fMRI Future Directions Conclusion

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9.1. INTRODUCTION The brain is a restless assembly of cells continuously working (i.e., receiving and routing information) to maintain its functional integrity. While advances in neuroscience (Kandel et al., 1991) have revealed that variations in neuronal activity – both electrical and chemical in nature – can affect the state of mind, mood, or behavior of the person, the importance of energetics as an indicator for neural function has been less appreciated. Energy is deﬁned as the capability of doing work and in a perfect nondissipating system the energy consumed equals the magnitude of work done (Garby and Larsen, 1995). The functioning brain is not far from equilibrium. Thus neurotransmission proceeds in a thermodynamically downhill fashion as a Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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consequence of transiently increased permeability of Na+ across the membrane down chemical gradients. Restoration of ionic gradients is energetically very expensive (see Chapter 7) because ions have to be transported against large chemical gradients (Creutzfeldt, 1975; Ames, 2000; Laughlin, 2001). Since most of the energy is spent to keep neurons ready to be able to work (i.e., ‘ﬁre’) whenever called for, measuring changes in energy use of a neuronal ensemble provides a window into their grouped activity. Under normal conditions, glucose is the main or exclusive carbon source for the adult mammalian brain and its oxidation is the main energy producing pathway (Siesjo, 1978). Glucose breakdown (through the tricarboxylic acid cycle) in the presence of oxygen generates ∼36 mol of ATP per mole of glucose, whereas in the absence of oxygen glycolysis and/or glycogenolysis generates 1–2 mol ATP per mole of glucose. Because complete glucose oxidation has a stoichiometry of C6 H12 O6 + 6O2 −−−→ 6CO2 + 6H2 O

(9.1)

the cerebral metabolic rate of oxygen consumption (CMRO2 ) is equal to six times the cerebral metabolic rate of glucose consumption (CMRglc ). Since results from experiments conducted under a variety of physiological conditions reveal that glucose oxidation is nearly complete, the relationship between CMRO2 and CMRglc is well established (Siesjo, 1978), and therefore well accepted (Shulman et al., 2001a). While small amounts of glucose can be stored as glycogen in the glia, there are no signiﬁcant stores of oxygen. Furthermore the volume of blood that provides the oxygenated hemoglobin – the main oxygen carrier in the brain – is extremely small. Because the brain in the average adult human has a very high energetic demand – approximately ten times that expected simply on the basis of its weight – even transient restrictions in oxygen supply can critically harm neural function (Sokoloff, 1991). Neuronal and glial activities are tightly correlated with their energy use and the brain’s energy reserve is not very signiﬁcant, so that a continuous circulation of blood – which provides nutrients and removes waste – is necessary for normal function. This phenomenon of neurovascular coupling is credited to Roy and Sherrington (1890) since they suggested, more than a century ago, a tight correlation between blood supply and cellular workload in brain tissue. Thus cerebral blood ﬂow (CBF) can provide an indirect estimate of the ongoing neural function under most conditions. Since most modern neuroimaging methods measure some component of CBF, the experimentalist’s inherent assumption has been that these maps reﬂect changes in neural energy, and hence alterations in neuronal activity. The extreme vulnerability of brain tissue to oxygen deprivation stipulates that fractional changes in CBF and CMRO2 as well as regional values of CBF and CMRO2 must effectively be correlated. An important point of consideration, however, is that although there is no requirement for a stoichiometric relationship between fractional changes in CBF and CMRO2 , there is a prescribed stoichiometric ratio between fractional changes in CMRO2 and CMRglc (i.e., Equation (9.1)) if glucose oxidation is to be maintained over a wide range of activity.

9.2. PHYSIOLOGY OF NEUROIMAGING The relationship between CMRO2 and CBF (i.e., fractional changes or regional values), and therefore mechanisms of oxygen transport from the blood to the brain, is critical for understanding neuroimaging (Hyder et al., 1998). A theory of substrate delivery from blood to tissue was formally introduced by Crone (1963). By detecting tracer amounts of an exogenous indicator’s dilution (within the blood) or diffusion (into the tissue) as a function of time, amounts of indicator permeating through the blood–brain barrier could be related to the arteriovenous concentration difference (i.e., Ca − Cv ) in a quantitative manner. The process of oxygen extraction by cerebral tissue from capillaries can be modeled almost the same way as the exogenous indicator’s dilution or diffusion curves because the only source of oxygen is the blood (i.e.,

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hemoglobin) and the major sink for oxygen is in the tissue (i.e., mitochondria). Thus a theoretical construct allowed CMRO2 and CBF to be related to Ca and Cv since oxygen extraction fraction (OEF) of the tissue is equal to (1 − Cv /Ca ) and to CMRO2 /(Ca CBF), according to Fick’s principle (Siesjo, 1978). CMRO2 = Ca − Cv CBF

(9.2)

The vital question about Equation (9.2) for neuroimaging is, however, what happens when there is increased neuronal activity? Two prior independent observations regarding the speciﬁcs of oxygenated blood and cerebral capillaries provided an insight. Hill (1913) identiﬁed that saturation of hemoglobin with oxygen, as reﬂected by the partial pressure of oxygen (pO2 ), changed the color of erythrocytes: the fully oxygenated form being bright red whereas the fully deoxygenated form being nearly black. Penﬁeld (1933) observed that arterial ends of cerebral capillaries were almost always red whereas venous ends were purple, and veins became redder when cellular workload was regionally increased. These independent observations provided a very important contention about Equation (9.2) and changes in local neuronal activity. In an activated region, which experiences a metabolically driven increase in local perfusion due to the augmented cellular work, the difference between Ca and Cv has to become smaller. Since the blood is almost fully oxygenated at the arterial end, the arteriovenous difference in capillaries can be decreased only if venous oxygenation is increased. Thus a metabolically driven increase in local perfusion is consistent with a drop in deoxyhemoglobin concentration at the venous end of the capillary. Therefore a qualitative understanding of functional hyperemia (i.e., synonymous with higher oxygenation), which has been known for several decades, suggests raised blood oxygenation with increased neuronal activity. In other words, a larger increase in CBF than CMRO2 , which in turn is commensurate with a decrease in OEF, would be expected in the activated region.

9.3. NEUROIMAGING WITH MRI Pauling and Coryell (1936) showed that deoxyhemoglobin is paramagnetic. Since hemoglobin is contained in erythrocytes, there are tiny magnetic ﬁelds in and around the cells. Because water protons can sense the local ﬁeld distortions around red blood cells, the changing amount of oxygen carried by hemoglobin could be sensed by NMR of water. Many decades later Ogawa et al. (1990) showed that a change of the venous deoxyhemoglobin concentration in brain capillaries can be detected in vivo with an MRI method (of water protons) which is sensitive to the microscopic magnetic susceptibility in the vasculature. Spin echo and gradient echo images are sensitive to functional hyperemia because these image contrasts can reﬂect physiologically induced changes in magnetic properties of blood. This new fMRI image contrast was therefore termed blood oxygenation level dependent (BOLD) by Ogawa et al. (1993). While the use of NMR physics can discern the BOLD image contrast with the measurable parameters of transverse relaxation time of tissue water (T2 SE and T2 GE with spin echo and gradient echo image contrasts, respectively), the physiological basis of interpreting fractional changes in fMRI signal (S/S) depends on hemodynamic and metabolic parameters because both CMRO2 and CBF can directly affect T2 SE or T2 GE in an MRI voxel. At steady state S/S is related to the metabolically driven increase in perfusion, S CBV CBF CMRO2 −N =M − (9.3) S CBF CMRO2 CBV where M and N are measurable physiologic and magnetic constants (related to T2 SE or T2 GE ), and CMRO2 /CMRO2 , CBF/CBF, and CBV/CBV are respective changes in oxidative metabolism, blood ﬂow and volume (for a review see Hyder et al., 2001). Note that M is equal to N divided by (1 + CBF/CBF). Obviously CMRO2 /CMRO2 is the most relevant of all of these physiological parameters

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for studying functional activity of the brain, because it is proportional to changes in energy use associated with alterations in neuronal activity induced by the stimulation. A conventional fMRI experiment offers information about where the neural activity has changed; however, a calibrated fMRI experiment – meaning that CMRO2 /CMRO2 is extracted from Equation (9.3) using measurements of CBF/CBF, CBV/CBV, and S/S – in addition presents valuable data about how much the neural activity has changed by. The main advantage of deriving CMRO2 /CMRO2 from calibrated fMRI, in contrast to 15 O PET (e.g., Mintun et al., 1984), 13 C MRS (e.g., Hyder et al., 1996), and 17 O MRS (e.g., Zhu et al., 2002) methods which can also measure CMRO2 /CMRO2 , is that the spatial and temporal resolutions are signiﬁcantly higher and it is an inexpensive and effortless method without exposure to radiation that can be conducted in conventional MRI scanners. In this chapter the energetic basis of BOLD image contrast (Figure 9.1(A)) is revealed from experiments conducted in rat brain at the high static magnetic ﬁeld strength (Bo ) of 7 T. The approach used can be summarized as follows. If S/S, CMRO2 /CMRO2 , CBF/CBF, and CBV/CBV are mapped (by different NMR methods) for a range of activity levels, the combined results can then be used to calibrate the BOLD signal (at a particular magnetic ﬁeld strength for a speciﬁc type of tissue) because CBF–CMRO2 (A) a

(B)

(C)

b Glucose + O2

Pre-synaptic neuron

T2 (baseline) < T2 (stimulation) O2 K+

ATP 300 × 300 × 1000 µm 220 × 220 × 500 µm Frontal cortex Parietal cortex (Axial slice) (Coronal slice) c

d

Na+

Na+ K

+

O2 220 × 220 × 250 µm 110 × 110 × 125 µm Olfactory bulb Olfactory bulb (Coronal slice) (Coronal slice)

Glucose + O2

Na+

O2

Capillary

Glucose + O2 ATP Na+

Baseline

Astrocyte Na+

ATP Capillary

Post-synaptic neuron

Stimulation

Figure 9.1. Neuroimaging with fMRI at 7 T. (A) Examples of localized activity detected with BOLD in anesthetized rat brain with forepaw stimulation (a, Hyder et al., 1994), single whisker stimulation (b, Yang et al., 1996), and olfactory stimulation (Yang et al. 1998) with high (c, Xu et al., 2000) and ultrahigh (d, Kida et al., 2002) spatial resolutions. (B) The fMRI signal changes indirectly reﬂect alterations in energetics of glutamatergic synapses (Shulman and Rothman, 1998; Attwell and Laughlin, 2001), where neuronal and astrocytic interactions are tightly regulated (Pellerin and Magistretti, 1994). A wide range of energy consuming processes (which include action potential propagation, maintenance of membrane potentials, vesicular recycling, neurotransmitter release and uptake) are involved in short-term neuronal information encoding, where the majority of the ATP utilized to support the cellular work is obtained from glucose oxidation. Since the only source of oxygen is hemoglobin in the blood and the major sink for oxygen is the mitochondria, a tight relationship between CBF and CMRO2 is mandated. (C) Because arterial blood in cerebral capillaries is fully oxygenated (Hill, 1913; Penﬁeld, 1933), the metabolically driven increase in local perfusion is such that the local increase in CBF is greater than the increase in CMRO2 (i.e., functional hyperemia). Thus an increased blood oxygenation is expected with neuronal activation which can be detected by spin echo (or gradient echo) MRI where T2 (or T2 ∗ ) of tissue water protons is increased thus causing an increase in the BOLD signal during the stimulation condition in comparison to the baseline condition. Since T2 (or T2 ∗ ) is directly affected by local magnetic susceptibility in an MRI voxel which can attenuate the MRI signal because of intravoxel spin dephasing, an increase in T2 (or T2 ∗ ) is commensurate with a lesser degree of intravoxel dephasing. The more oxygenated red blood cell is shown in lighter gray, the arterial and venous ends are shown by the two arrows pointing in and out of the capillary, respectively.

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and CBF–CBV relationships can be quantiﬁed to determine the constants in Equation (9.3) (i.e., M and N ). Since a majority of energy consumed is spent in restoring ionic gradients across the cell membrane to maintain functional integrity (Figure 9.1(B)), demand and supply of nutrients are tightly correlated. Thus CMRO2 values provide insight into the bioelectric work which helps sustain excitability of neurons. The obligatory dilatation of microvasculature (Figure 9.1(C)), which is the mechanical response that was noticed by Roy and Sherrington (1890), is involved in removal of waste (e.g., CO2 , heat) while providing nutrients and is reﬂected in the CBF–CBV relationship. The type of BOLD calibration described here is unique since it adds a measure of changes in energy consumption, CMRO2 /CMRO2 , to the valuable localization results generally considered.

9.4. BIOPHYSICS OF fMRI There has been great progress in the biophysical understanding of contrast enhancement with MRI contrast agents such as gadolinium (e.g., Haacke et al., 2001). Thus the theory of susceptibility-based contrast mechanisms for an endogenous paramagnetic material such as deoxyhemoglobin is becoming well understood (Albert et al., 1993; Kennan et al., 1994; Weisskoff et al., 1994). While a detailed discussion of this topic is not the objective here, some key elements are mentioned to understand the role of cerebral microvasculature in the BOLD image contrast. The presence of deoxyhemoglobin, behaving as an endogenous contrast agent, within a uniform cylinder of inﬁnite length creates local magnetic ﬁeld gradients which extend into the surrounding tissue and dephase water protons. These local gradients are a consequence of the volume magnetic susceptibility difference between tissue and the compartmentalized paramagnetic material inside the capillary. For some randomly oriented cylinders, which mimic the capillary network within tissues, the local gradients in the vicinity of proximal capillaries overlap (provided that capillaries are far enough from each other to discount diffusion of water from one vessel to another). The local gradients outside the capillaries enhance the dephasing of water protons (i.e., spins) in the tissue which can be detected by decreased signals in both T2 SE and T2 GE weighted images. For functional hyperemia, of course, the amount of deoxyhemoglobin is decreased and the entire argument above is reversed (Figure 9.2(A)). Since the difference between Ca and Cv becomes smaller with functional hyperemia due to increases in Cv (see Equation (9.2)), the contributions of arterial blood can be disregarded because arterial blood is almost fully oxygenated (Hill, 1913) and it is diamagnetic (Pauling and Coryell, 1936). Because studies of cerebral vascular anatomy reveal that the arteriole blood volume (as well as the surface area) is a small fraction of the entire blood volume (Craigie, 1945) in cerebral microvasculature (Figure 9.2(B)), the neglected arteriole compartment in BOLD image contrast does not present a signiﬁcant bias. How are spin echo and gradient echo image contrasts affected by local ﬁeld gradients that are generated by the compartmentalized paramagnetic material inside capillaries? The diffusion of spins in the tissue space through paths that contain the perturbing ﬁelds subjects the spin ensemble to an averaged ﬁeld distribution. While BOLD image contrast does not measure spin diffusion directly, a suitable choice of echo time (TE) sensitizes the experiment to the speed of diffusion (i.e., fast, intermediate, and slow). In all regimes, the diffusive effects introduce a ‘dynamic’ averaging of local ﬁeld gradients which causes signal decay and this loss of spin coherence is not reversible. However, in addition there are also static effects which introduce a ‘local’ averaging of the ﬁeld gradients but this signal decay is reversible (e.g., by a refocusing pulse in a spin echo sequence). Both static and diffusive effects are present evenly in the intermediate regime (e.g., outside blood vessels, diameter 5–15 µm) and either image contrast is sensitive. In fMRI it is the regions with fast and intermediate diffusion that are of interest because they reﬂect signals from tissue outside of small blood vessels (for a recent review see Ugurbil et al., 2000). The contrast enhancement by deoxyhemoglobin for mapping neural activity should speciﬁcally respond to small vessel effects. There are two types of vascular effects, types A and B. Since the blood volume

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(D)

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(C)

b=0 Low field

High field

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Extra Intra

Intra TE (ms)

b = 400

TE (ms)

Figure 9.2. The biophysical basis of fMRI. (A) Since deoxyhemoglobin is paramagnetic (Pauling and Coryell, 1936), the presence of this endogenous contrast agent inside cerebral capillaries generates local magnetic ﬁeld gradients which extend into the tissue (Ogawa et al., 1990). The local gradients enhance dephasing of water protons in the tissue which can be detected by decreased signals in T2 SE and T2 GE weighted images. During activation the amount of deoxyhemoglobin is decreased in comparison to the resting condition and this difference between the stimulation-induced susceptibility difference of blood can be observed as changes in T2 (or T2 ∗ ) by spin echo (or gradient echo) MRI. (B) Since the arteriole blood volume and surface area are signiﬁcantly smaller than other compartments of cerebral vasculature (Craigie, 1945), the neglect of the arteriole compartment in BOLD image contrast does not provide an incredible bias. (C) At any given Bo , the intravascular and extravascular weightings in BOLD data depend on the transverse relaxation times of water in blood (T2 intra ; black) and tissue (T2 extra ; gray). Prior studies (for a recent review see Ugurbil et al., 2000) have shown that T2 intra ≈ T2 extra at low Bo which suggests that both intravascular and extravascular compartments are equally dominant, whereas measured values show that T2 intra < T2 extra at high Bo which indicates that the extravascular compartment is far more dominant. In each case, the intravascular and extravascular fractions are shown by the dotted lines. (D) The contribution of the intravascular effect on BOLD can be examined by using graded diffusion-weighted spin echo MRI (e.g., Lee et al., 1999) with a range of diffusion weighting factor (b) values – the higher the b value the greater the degree of suppression of the intravascular contribution to the BOLD effect. The ﬁgure shows two spin echo fMRI maps (TE = 50 ms) of the forepaw stimulation model of the same rat brain at 7 T with b values of 0 and 400 s/mm2 . The high correlation between these two maps suggests negligible intravascular contamination of the BOLD effect at 7 T.

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fraction tends to be small (2–5 %) for a typical MRI voxel, there is an inherent partial volume bias towards signal from tissue water with either contrast. However, very large vessels (e.g., arteries and veins) can have larger partial volume and these ‘false positives’ due to type A vascular effect in gradient echo data can be removed by comparison with spin echo data. Furthermore it is important to realize that intravascular and extravascular weightings in BOLD data depend on the actual values of transverse relaxation times of water in blood (T2 intra ) and tissue (T2 extra ). Because T2 intra is slightly different from T2 extra at low Bo the signal is equally weighted by both compartments (Figure 9.2(C)), whereas at higher Bo the signal is more weighted by the extravascular compartment since T2 intra is signiﬁcantly shorter than T2 extra (Figure 9.2(C)). This type B vascular effect can be compressed by diffusion gradients in spin echo MRI (Boxerman et al., 1995; Song et al., 1996; Zhong et al., 1998; Lee et al., 1999) to reduce signals of slow and intermediate diffusion (Figure 9.2(D)). Although the BOLD effect at Bo of 4 T and higher depicted by spin echo and gradient echo data is predominantly associated with the small vessel effects, the gradient echo data may partly be affected by vascular artifacts (for a recent review see Ogawa et al., 1998). While high Bo fMRI with diffusionweighted spin echo is the ideal image contrast for neuroimaging (Figure 9.2(D)), the concern of enormous sensitivity losses makes this method less preferred. Instead the use of both spin echo and gradient echo image contrasts have proven to be more useful (for a recent review see Hyder et al., 2001). In both image contrasts there is a relaxation term (T2 o ) that is unrelated to susceptibility based effects (i.e., dipole–dipole), 1 T2GE 1 T2SE

=

1 1 1 1 + Bo o + + T2 T2 T2 T2

(9.4a)

=

1 1 + T2 T2o

(9.4b)

where T2 Bo is the component attributed to local Bo inhomogeneity (Bo ), and T2 and T2 are the reversible and nonreversible terms due to blood oxygenation effects. The BOLD calibration method shown here removes the unknown component T2 o and in addition the T2 Bo term (see Equation (9.5)). The result is a term that is purely dependent on blood oxygenation effects around small blood vessels and devoid of all other relaxation terms (Equations (9.5) and (9.6)). Although the comparative sensitivity of the BOLD effect detected by this calibration approach is lower because the fraction of measured change in T2 SE or T2 GE that is used for localizing activated regions is smaller (see Equation (9.4)), the combined use of both T2 SE and T2 GE maps provides a much better statistical basis for localization of weaker activated regions.

9.5. METHODS FOR CALIBRATING fMRI The approach used to calibrate BOLD as described here requires quantitative relationships between physiological parameters (CBV, CBF, and CMRO2 ) and BOLD signal (S) to be established, under physiological conditions. In contrast, previous studies have measured at best two of these parameters and assumed the other parameters under conditions of nonphysiologic perturbations (e.g., hypoxia or hypercapnia). Since the physiologic perturbations used in this type of BOLD calibration (Kida et al., 2000; Hyder et al., 2001) were within the autoregulatory range (all of which conserved similar CBF–CMRO2 and CBF–CBV relationships expected from functional hyperemia), results from the rat brain can be used for guiding comparable BOLD calibration experiments in humans. While the idea of this type of BOLD calibration is quite simple (i.e., measure changes in all parameters in Equation (9.3) induced by physiologic perturbations which are well within the autoregulatory range so that constants M and N are used without complications), implementation is quite difﬁcult because of methodological concerns. There are two main reasons why this type

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of BOLD calibration approach has not been favored in other laboratories: ﬁrst, to measure quantitative changes in S, CBF, CBV, and CMRO2 both high resolution MRI and MRS methods are necessary; second, only physiologic perturbations that induce simultaneous changes in all parameters in Equation (9.3) can be used because conventionally used perturbations in MRI experiments (e.g., hypoxia, hypercapnia) are inadequate since the perturbations induce different effects in comparison to functional hyperemia (e.g., ‘uncoupling’ of CMRO2 and CBF). An additional beneﬁt of this type of BOLD calibration is that while CMRO2 /CMRO2 is extracted from Equation (9.3), the prediction can be validated by comparison with measured values. All methods described below were applied in anesthetized rats (systemic physiologic parameters were monitored and controlled) on a 7 T horizontal bore system using a radio frequency probe for both 1 H/13 C MRS and 1 H MRI experiments with an arrangement of two orthogonally oriented 1 H coils (surface receiver, volume transmitter) which permitted better local shimming and higher sensitivity (via homogeneous transmission and minimum loss in surface reception). There were a total of ﬁve steady state treatment groups (i.e., α to ε) for the multimodal measurements. The rats in group α were anesthetized with morphine (∼65 mg/kg/h), whereas in groups β and γ the rats received in addition pentobarbital (∼30 mg/kg/h) and nicotine (∼10 mg/kg/h) respectively to decrease and increase global activity from baseline. The rats in group δ were anesthetized with α-chloralose (∼60 mg/kg/h), whereas the rats in group ε (i.e., α-chloralose anesthesia) received in addition electrical forepaw stimulation (3 Hz) to increase activity from the baseline. The different methods (see below) were used to obtain the multimodal data on the same rat. Spin echo and gradient echo MRI sequences were used to measure fractional changes in BOLD signal, S/S. Since the degree of T2 SE and T2 GE weighting in spin echo and gradient echo data can be varied by changing TE, multiple TE values (10 – 80 ms) were used to obtain absolute T2 SE and T2 GE maps, respectively. The multiple TE data were rapidly acquired with echo planar imaging (EPI) and singleexponential ﬁts were used to calculate the T2 SE and T2 GE maps, where the latter contained the T2 Bo relaxation component (see Equation (9.4)). The T2 Bo component within the region of interest is measurable with an MRI spin tagging method (Mosher and Smith, 1991), and therefore removable within a range of accuracy. The resolution of this ﬁeld inhomogeneity estimate is on the order of 1.0 mm2 (i.e., coarser than the 0.2 mm2 in-plane MRI resolution). Since the biophysical basis of T2 SE and T2 GE shows that there are overlapping terms (see Equation (9.4)), the difference of these two relaxation terms after removal of the T2 Bo component 1 1 1 = GE − SE (9.5) T2 T2 T2 exposes the T2 which is the reversible term that is due to pure blood oxygenation effects. The assumption in Equation (9.5) is that T2 SE and T2 GE contain the same nonreversible relaxation component, which is also dependent on blood oxygenation, and by differencing of 1/T2 SE and 1/T2 GE the remaining term is devoid of relaxation terms related to nonsusceptibility based effects. Since 1/T2 was originally described by Ogawa et al. (1993) to be related to venous deoxyhemoglobin as well as the blood volume in the voxel, the fractional change in BOLD signal in Equation (9.3) is also given by S 1 × TE − 1 = exp − S T2

(9.6)

where (1/T2 ) is the change in T2 due to functional hyperemia. Changes in CBV were measured by administration of a high susceptibility (iron oxide) MRI contrast agent which has a long half-life due to an extremely slow clearance rate (Bauer and Schulten, 1992; Jung and Jacobs, 1995). Since the presence of the exogenous contrast material within the microvasculature

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enhances the blood volume induced changes in T2 SE or T2 GE , the same MRI methods (as described above) can be used for CBV measurements. The relative changes in CBV were calculated by (Kennan et al. (1998)) R w − R w/o CBV = CBV R w − R w/o

(9.7)

where R w and R w/o are 1/T2 SE (or 1/T2 GE ) at baseline conditions with and without agents respectively, and R w and R w/o are absolute differences in 1/T2 SE (or 1/T2 GE ) due to the perturbation with and without the contrast agents, respectively. Equation (9.7) is based on the assumption that changes in T2 SE or T2 GE are separable in terms of changes in blood volume and blood oxygenation, as long as blood hematocrit effects are not signiﬁcant enough to warrant inclusion (Mandeville et al., 1998). Since the long half-life of the high susceptibility or super-paramagnetic MRI contrast agents arises from preferential compartmentation in the microvasculature by uniform distribution within blood plasma, the derived changes in CBV therefore mainly reﬂect changes in plasma volume (van Bruggen et al., 1998). However, under the assumption of constant hematocrit the changes in plasma and hemoglobin in the microvasculature would be equivalent (Kennan et al., 1998). Absolute CBF maps were imaged by using magnetically labeled arterial blood water which acts as an endogenous perfusion tracer, the kinetics of which can be determined by changes in the longitudinal relaxation time of tissue water (T1 ) obtained by inversion recovery methods (Detre et al., 1992; Williams et al., 1992; Zhang et al., 1992). The primary assumption with this MRI method (Calamante et al., 1999), much like its counterpart from H2 15 O PET (Raichle et al., 1983), is that water is a freely diffusible tracer (Eichling et al., 1974; Paulson et al., 1977; Silva et al., 1997). Thus when water in the blood is tagged and the tracer is then followed into the tissue, water perfusion through the tissue can be inferred. The conventionally measured T1 map used for anatomical imaging, which is basically insensitive to perfusion, is obtained by global inversion recovery (i.e., both blood and tissue water are inverted nonpreferentially). Similarly, an additional T1 map can be obtained with a more localized inversion (or saturation) of spins (T1 app ) that makes the contrast more sensitive to perfusion because the blood water can be selectively inverted (or saturated). The degree of loss of the tracer from the blood, the fate of which is mainly the tissue, is related to perfusion and can be inferred from comparison of the ‘unlabeled’ and ‘labeled’ T1 maps, respectively, with global and local inversions, 1 CBF × 1 = app + T1 λ T1

(9.8)

where λ is the assumed brain–blood partition coefﬁcient of 0.95 ml/g and is a correction factor that accounts for the difference between relaxation of tissue water and arterial blood water (as well as units of ml/g/min for CBF). Absolute CBF maps were obtained by using combination of slice selective and global inversion recovery weighted EPI sequence and single exponential recovery ﬁts to the multi inversion recovery data (200–2200 ms). Because the approach used here relied on comparison of T1 maps (Schwarzbauer et al., 1996), arterial transit time artifacts associated with differencing ‘unlabeled’ and ‘labeled’ maps of a particular inversion recovery time could be ignored. Since a large homogenous radio frequency transmission coil (covering the rat’s torso) was used for inversions, there was negligible contamination of ‘untagged’ blood volume in the T1 maps (Hyder et al., 2000a). Other artifacts were minimized with optimal choice of inversion and excitation slice thickness ratio, adiabatic inversion pulses, short inversion pulse durations, and image acquisition under fully relaxed conditions (Hyder et al., 2000b). Absolute values of CMRO2 were obtained by using indirect 13 C MRS (i.e., detection of 13 C with sensitivity of 1 H with the proton observe carbon edit (POCE) method (Rothman et al., 1985; Fitzpatrick et al., 1990; Hyder et al., 1996; Pfeuffer et al., 1999; Henry et al., 2000; Chen et al., 2001; de Graaf et al., 2003))

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in conjunction with substrates labeled with the 13 C isotope which have been used in vivo to study metabolic ﬂuxes by detecting turnover of the 13 C isotope into other pools (for recent reviews see Rothman et al., 1999, 2002, 2003). The reader is directed towards Chapters 3 and 4 regarding the details of 13 C MRS methods and modeling of the 13 C turnover data. Thus 13 C MRS provides a unique window on metabolism of 13 C-labeled glucose which is the major energy substrate, under normal conditions (see Chapters 2 and 5). In contrast to the radioactive 14 C isotope detected with 2-deoxyglucose autoradiography (used to measure CMRglc ), the nonradioactive 13 C isotope in conjunction with 13 C MRS measures one or more metabolites at different carbon positions in real time. In each case, the spatial localization of each POCE study (8–48 µl) (Hyder et al., 1996, 1997, 1999, 2000b) was guided by previously acquired CBF maps. Glutamate and γ -amino butyric acid (GABA) are the major excitatory and inhibitory neurotransmitters, respectively, and play dominant roles in mediating brain activity. In these POCE studies the 13 C labeling of C4-glutamate was measured during infusion of [1-13 C] or [1,6-13 C] glucose to calculate tricarboxylic acid cycle ﬂux (VTCA ) which is stoichiometrically linked to CMRO2 , 3 CMRO2 = 3VTCA + Vdil 4

(9.9)

where Vdil accounts for measurable 12 C inﬂows from other substrates (e.g., ketone bodies) which can dilute the 13 C fractional enrichment of the acetyl CoA pool. Thus the POCE method provides an estimate of the energetic costs associated with glutamatergic neurotransmission (see Chapter 5), with small but measurable corrections of the GABAergic system and glial oxidation (see Chapter 6). Several modeling parameters have secondary effects upon the calculated ﬂuxes, and the magnitudes of these effects have been evaluated by sensitivity analyses and in vivo experiments (see Chapter 4). Since these studies reveal that these parameters have relatively small effects on VTCA (±5 %) for the range covered in anesthetized rat studies, the derived ﬂuxes with [1-13 C] or [1,6-13 C] glucose provide valuable information on neuroenergetics.

9.6. PHYSIOLOGICAL BASIS OF fMRI An example of the multimodal methods described above applied to rat brain imaging in vivo is shown in Figure 9.3 for a particular baseline condition. This type of multimodal data spanning a range of activity levels provided insight into the CBF–CMRO2 and CBF–CBV relationships that form the physiological basis of BOLD. For a region of interest in the somatosensory cortex, Figure 9.4(A) shows that for a speciﬁc magnitude of change in CBF from baseline, the change in CMRO2 far exceeds the CBV change. This indicates that the BOLD signal is inﬂuenced strongly by the main energy consuming processes and the depiction of BOLD signal simply as mechanical events (of dilating microvessels) ignores a signiﬁcant physiologic makeup of the image contrast. The constants M and N in Equation (9.3) can be estimated using the measured CBF–CMRO2 and CBF–CBV relationships along with commensurate changes in the BOLD signal (Figure 9.4(B)). The constants are apt for a baseline predeﬁned by the range of activities covered and corresponding distribution of T2 values (see Figure 9.5). Since only physiological perturbations were used for the BOLD calibration, the near-linear relationship between MRI-measured CBF and POCE-measured CMRO2 over the entire range of activity covered was expected (Figure 9.5(A)). However, the range of the 1/T2 values (see Equation (9.5)) was grouped over a speciﬁc range of CMRO2 and CBF values, as shown in the inset to Figure 9.5(A). Thus the corresponding distributions of CBF and/or CMRO2 values were used to determine the predeﬁned baseline condition (boxed region in Figure 9.5(A)) for which the derived constants M and N were valid. Support for this type of BOLD calibration comes from comparison of NMR-measured absolute values of CBF and CMRO2 values (in this laboratory) and measurements under similar conditions in rat brain by other methods (including

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the 17 O MRS method discussed in Chapter 8). As shown in Figure 9.5(B), the agreement from different methods for CBF and CMRO2 measured in the rat brain is excellent (Table 9.1). Using the experimentally derived CBF–CMRO2 and CBF–CBV relationships along with changes in the BOLD signal (Figure 9.4), the constants M and N were used with Equation (9.3) along with measured values of CBF/CBF, CBV/CBV, and S/S to predict the changes in CMRO2 , as shown in Figure 9.6(A). Because most of the data points comparing the predicted (from calibrated BOLD) and measured (from localized POCE) values of CMRO2 /CMRO2 fall close to the line of identity over a wide activity range, the BOLD calibration was accurate within 15 % (Kida et al., 2000; Hyder et al., 2001). The validation of the calibration thus allowed the prediction of CMRO2 /CMRO2 for functional activation studies in rat brain at 7 T, as shown in Figure 9.6(B), where changes in S, CBF, and CBV were measured in the same rat. Although the localized changes in the different parameters were quite well correlated in the example shown (Figure 9.6(B)), it is important to note that a voxel of high intensity in the BOLD map did not always correspond to a high intensity voxel in the CMRO2 map (data not shown). These subtle variations arise from the activity variations of different parameters throughout the regions of interest, which when included with the calibration provides a constrained and scaled representation of CMRO2 changes. A likely source of uncertainty in comparing the MRI-predicted CMRO2 data with the POCE-measured CMRO2 data (Figure 9.6(A)) is that the heterogeneity in metabolism within the larger MRS voxel (in the range of µl) in comparison to the multimodal MRI data (of CBF/CBF, CBV/CBV, S/S), which have substantially higher spatial resolution (in the range of nl). Furthermore the degree of heterogeneity is greater under conditions of stimulation-induced activity (i.e., from group δ to ε) than pharmacologically induced activity (i.e., from group α to γ ) in a particular region, based on CMRglc data from 2-14 C-deoxyglucose autoradiography studies in anesthetized rats (Ueki et al., 1988). These transitions are graphically shown in

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Figure 9.4. Summary of quantitative MRI and MRS measurements of relative changes in CBF, CMRO2 , CBV, and S over a wide range of neuronal activity. (A) Comparisons of CBV/CBV ( ) and CMRO2 /CMRO2 (×) versus CBF/CBF for the different conditions (groups α – ε). The groups β and ε are located at the extreme left and right corners, respectively. The group δ is immediately next to group β. The group γ is located left of the origin, whereas the group α is located left of group γ . The ratios of (CMRO2 /CMRO2 )/(CBF/CBF) and (CBV/CBV)/(CBF/CBF) are signiﬁcantly different, where the slopes through the origin are ∼0.7 and ∼0.1, respectively (Kida et al., 2000; Hyder et al., 2001). The origin represents the resting awake state for rat cerebral cortex (Nilsson and Siesjo, 1975). (B) The changes in CMRO2 , CBV, and S were normalized by changes in CBF. This normalization procedure provides a comparison of relative changes in each parameter during changes in neuronal activity. These ﬁndings show that CBF and CMRO2 play a more dominant role in modulation of BOLD signal at 7 T in glutamatergic neurons of rat brain. The error bars represent the standard deviation from the mean.

Ž

Figures 9.4(A) and 9.5(A). Since the CMRO2 measured (by localized POCE) in the larger MRS voxel is equivalent to the averaged CMRO2 predicted (by calibrated BOLD) from all the MRI voxels in a particular region, the voxels at the MRI spatial resolution with either very high or very low CMRO2 values (which cannot reliably be characterized by the current BOLD calibration) would have the greatest level of uncertainty. An important aspect of this type of BOLD calibration, however, is that both increase and decrease of activities were examined for the changes in the BOLD signal (e.g., transition from group α to β was a decrement, whereas transition from group α to γ was an increment, as shown in Figures 9.4(A) and 9.5(A)). Therefore, the constants M and N can be used conﬁdently, within the experimental errors of the methods used as well as the accuracy of the BOLD calibration, to determine both decrements and increments of CMRO2 throughout the somatosensory cortex provided that the perturbations are physiological in nature. Since changes in both CMRO2 and CBV (in relation to CBF) can affect the BOLD signal, it is important to compare the magnitudes of change in each parameter with previous measurements. However, it is known that the magnitude of CMRO2 /CMRO2 (or CMRglc /CMRglc , or CBF/CBF, or CBV/CBV) is dependent on resting baseline values which in turn are contingent on the depth of anesthesia (for recent reviews see Shulman et al., 1999, 2001b, 2002, 2003). Instead, comparing the magnitudes of (CMRO2 /CMRO2 )/(CBF/CBF), (CMRglc /CMRglc )/(CBF/CBF), and (CBV/CBV)/(CBF/CBF) can help negate the variable effects of anesthesia for functional activation studies in animals. The values of CMRO2 /CMRO2 predicted by BOLD (in Figure 9.6(B)) cannot be directly compared with previous measurements of CMRO2 /CMRO2 (POCE) and CMRglc /CMRglc (2-14 C-deoxyglucose) because the anesthetic dosages were different in the BOLD study. However, ratios of (CMRO2 /CMRO2 )/(CBF/CBF) and

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Figure 9.5. Absolute values of CMRO2 and CBF in the rat somatosensory cortex. (A) Measurements of CMRO2 and CBF in the same rat by 13 C MRS and MRI methods for the ﬁve levels of activities (groups α – ε) measured in this laboratory (Hyder et al., 2000b). The inset shows the distribution of 1/T2 values for the same conditions as a function of the ratio of CMRO2 /CBF values (Hyder et al., 2001). Since the 1/T2 values were clustered around a range of CMRO2 /CBF values (shown by the gray square), the calibration of the BOLD effect is biased towards this predeﬁned baseline condition. (B) Measurements of CMRO2 and CBF in the rat from other laboratories (black symbols) are compared with measurements from this laboratory (gray circles). The dotted lines represent the nonanesthetized awake condition in rat brain (Nilsson and Siesjo, 1975). The details of the square symbols are shown in Table 9.1 whereas the triangle symbol represent measurements from Zhu et al. (2002) using 17 O MRS. Table 9.1.

CBF and CMRO2 of rat brain measured in other laboratories

Reference Nilsson and Siesjo (1975) Nilsson and Siesjo (1975) Hagerdal et al. (1975) Nilsson and Siesjo (1976) Nilsson and Siesjo (1975) Nilsson and Siesjo (1975) Zhu et al. (2002)

Condition

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Conscious (awake) Phenobarbital (50 mg/kg) 70 % N2 O 70 % N2 O+ 1.2 % halothane Phenobarbital (150 mg/kg) Phenobarbital (250 mg/kg) α-Chloralose (45 mg/kg/h)

1.4 1.1 1.1 0.8 0.8 0.6 0.5

5.0 4.1 4.6 3.4 3.0 2.6 2.2

(CMRglc /CMRglc )/(CBF/CBF) from all of these studies (Ueki et al., 1988; Grunwald et al., 1987, 1991; Otsuka et al., 1991a,b) are reasonably comparable (∼0.7), which supports the belief that oxidative glycolysis provides most of the energy during functional activation. Likewise, the ratio of (CBV/CBV)/(CBF/ CBF) measured with superparamagnetic contrast agents is also in good agreement (∼0.1) with prior results for the same rat model (van Bruggen et al., 1998; Silva et al., 1999). However, the (CBV/CBV)/(CBF/ CBF) ratio may not be the same for other nonphysiologic perturbations. Another approach that has been used for BOLD calibration studies incorporates a CO2 challenge in conjunction with the ‘normal’ experiment (i.e., a functional challenge). The ﬁrst assumption in this type of BOLD calibration is that the CBF–CBV relationship is the same in both perturbations. The second

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Figure 9.6. Validation of calibrated fMRI. (A) Comparison of predicted (by calibrated fMRI) and measured (by 13 C MRS) values of CMRO2 /CMRO2 provide validation of the BOLD calibration at 7 T (Kida et al., 2000; Hyder et al., 2001). The dotted line represents the line of identity. These results indicate that at high Bo the microscopic susceptibility effects of deoxygenated hemoglobin dominates the relaxation effects in tissue, which can be accentuated by combining the gradient echo and spin echo data (Equation (9.5)). Since the validation of BOLD calibration relies on standard errors of independent measures (for relaxation rate, blood ﬂow, and volume), by comparing calculated and measured values of CMRO2 /CMRO2 it can be calculated that the validation accuracy for high resolution CMRO2 mapping by multimodal MRI at 7 T in rat cortex is at least 80 %. (B) Multimodal maps of localized changes in CBF, CBV, and S (Equation (9.3)) obtained during forepaw stimulation in an α-chloralose-anesthetized rat which were used to calculate a relative CMRO2 map. The changes in CBF, CBV, and S were 1.17 ± 0.41, 0.07 ± 0.04, and 0.06 ± 0.02, respectively, and the localized change in CMRO2 was 0.93 ± 0.33. The regional correlation observed between changes in CMRO2 and CBF is very similar to regional correlation between changes in CMRglc and CBF for the same rat model (Ueki et al., 1988).

assumption is that changes in CMRO2 (i.e., neuronal activity) during a CO2 challenge are negligible, although with prolonged exposure to high CO2 levels various pools in the tricarboxylic acid cycle become depleted (Siesjo, 1978). In most of these prior studies (van Zijl et al., 1998; Davis et al., 1998; Hoge et al., 1999; Kim et al., 1999) the changes in CBV were not measured at all and assumed a dependence on changes in CBF, CBV CBF φ = 1+ −1 (9.10) CBV CBF where φ was determined to be ∼0.4 from a PET study of hypercapnic challenge in primates (Grubb et al., 1974). In contrast, the CBF–CBV relationship derived from the MRI studies (Figure 9.4(A)) shows that the value of φ is actually closer to ∼0.1 in the rat brain. The implication of using this different value for φ is that when this parameter is overestimated, as in some recent human fMRI studies (see below) because CBV changes cannot be measured directly due to lack of an approved MRI contrast agent, the changes in CMRO2 would be underestimated (Equation (9.3)). While this discrepancy of the CBF–CBV relationship (i.e., differing values of φ) can be qualitatively explained by the facts that distribution spaces and half-lives of PET (Ito et al., 2001, 2003) and MRI tracers (Kida et al., 1999, 2000) are different, a greater concern is that a BOLD calibration experiment for a hypercapnia or a hypoxia challenge is only valid for similar perturbations because both the blood pH and hematocrit are signiﬁcantly altered (Siesjo, 1978) in comparison to normal physiological conditions.

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Recently there has been a lot of interest in exploring the neural basis of fMRI by combining electrophysiological methods (Yang et al., 1997; Brinker et al., 1999; Ogawa et al., 2000; Rees et al., 2000; Logothetis et al., 2001). The correlations that have been observed between BOLD and electrical signals from neurons are based on aspects of establishing spatial overlap and matching temporal dynamics. Despite large disparities in the temporal dynamics as well as spatial responses of each signal type, these correlations are delicate at best because an experimentalist still cannot quantitatively infer from the fMRI data the degree by which the neural activity has changed in an activated focus. An unconquerable limitation of fMRI will always be that even a voxel of ultrahigh spatial resolution (e.g., 0.001 µl voxel in the rat olfactory bulb in Figure 9.1(A)) will contain at least several thousand neurons. Thus at best a BOLD signal when calibrated may be used to reﬂect the energetics of an ensemble of neurons (for a recent review see Hyder et al., 2002). Since energy is deﬁned as the capability of doing work, how efﬁciently do brain cells utilize energy for function? Extracellular recordings were used to measure the spiking frequency of an ensemble of neurons (ν) contained in a fraction of an fMRI voxel (Smith et al., 2002). Since ν is a reliable and quantiﬁable measure of brain work, changes in this parameter with different workloads would reﬂect the changing degrees of work done by the ensemble. The results show that fractional changes in energy (CMRO2 /CMRO2 ) were approximately equal to fractional changes in spiking rate (ν/ν) during stimulations from different baseline conditions (Figure 9.7(A)), which show that particular changes in neural energy expended approximately equal percentage changes in work done in a localized region. Thus calibrated fMRI used to obtain CMRO2 can provide a view of the ensemble work in a voxel. The details of the experimental approach and their implications are discussed in Chapter 10.

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Figure 9.7. Neural basis of fMRI. (A) In the α-chloralose-anesthetized rat, forepaw stimulation-induced changes in neuroenergetics (CMRO2 /CMRO2 ; by calibrated fMRI) and spiking rate of a neural ensemble (ν/ν; by extracellular recordings) were measured from the somatosensory cortex (Smith et al., 2002). Since CMRO2 /CMRO2 ≈ ν/ν over a wide range of activity in rat brain, the changes in neural energy is commensurate with changes in neural work done in a local region (Hyder et al., 2002). Thus CMRO2 /CMRO2 obtained from calibrated fMRI may provide insight into grouped activity a neuronal ensemble in a voxel. (B) Lamotrigine, which attenuates activity of pre-synaptic voltage-dependent Na+ channels and glutamate release (Leach et al., 1986), was used to illustrate the involvement of voltage-dependent Na+ channels and glutamate release on BOLD signal. The ﬁgure shows the localized BOLD responses during forepaw stimulation in an α-chloralose-anesthetized rat with (+) and without (−) lamotrigine (Kida et al., 2001), at the same statistical threshold. Since only the stimulus-evoked BOLD (and CBF) responses were depressed with lamotrigine treatment, the voltage-dependent Na+ channels may play a signiﬁcant role in evoked neurovascular responses, whereas under anesthesia the baseline would depend on action of voltage-independent Na+ channels.

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To date much of fMRI research pertains to measurements of activity-dependent changes in physiological parameters mentioned above. It is also important to characterize the molecular mechanisms linking the BOLD signal to neurophysiological events. Under physiological conditions Ca2+ -dependent vesicular release of neurotransmitters (e.g., an excitatory amino acid such as glutamate) occurs in response to depolarization which results from inﬂux of Na+ ions through pre-synaptic voltage-dependent Na+ channels. Neuronal excitability and neurotransmitter release can both be suppressed by inhibitors of these channels (e.g., lamotrigine) as well as the Na+ current that these channels mediate (Figure 9.1(B)). Is activation of voltagedependent Na+ channels a necessary step in the neurochemical pathway leading to the BOLD signal change as well as the local CBF response during functional activation? The stimulus-evoked BOLD and CBF responses (i.e., energetics) were signiﬁcantly depressed following lamotrigine treatment (Kida et al., 2001). Lamotrigine – a drug that is commonly used in treatment of epilepsy – speciﬁcally attenuates the activity of the pre-synaptically located voltage-dependent Na+ channels (Leach et al., 1986). However, there is a signiﬁcant fraction of Na+ channels that are voltage independent (Kandel et al., 1991). In fMRI studies of animal the baseline is largely maintained by anesthesia which presumably affects all Na+ channels. Lamotrigine mainly suppresses the evoked neurovascular response, while basal activity remained unchanged (Figure 9.7(B)). This implies that the voltage-dependent Na+ channels play a major role in the evoked neurovascular response, whereas under anesthesia the baseline depends on the action of voltage-independent Na+ channels. The measured proportionality between CBF and CMRO2 in anesthetized rats (Figure 9.5) supports the view that anesthetics affect the voltage-independent Na+ channels (Lees, 1972), although exact mechanisms of anesthetic action are unknown (Richard, 1998).

9.8. IMPLICATIONS FOR HUMAN fMRI The results from anesthetized rats have consequences for awake human fMRI studies. Although BOLD calibration experiments of this type have not been performed in awake humans yet, some well-informed Table 9.2. Changes in CMRO2 , CMRglc , and CBF of awake human brain measured by PET in different laboratories for a range of sensory stimulations. Numbers in italics represent averaged values. See Figure 9.8 and Rothman et al. (2002) for implications CBF CBF 0.30 0.28 0.31 0.18 0.18 0.18 0.27 0.43 0.49 0.25 0.26 0.32 0.38 0.42 0.13 0.88

CMRO2 CMRO2 0.40 0.09 0.13 −0.03 −0.01 0.13 0.13 0.13 0.05 0.28 0.29 0.10 0.16 0.06 0.12 0.47

CMRglc CMRglc

Reference

0.21 0.17 0.18 0.08 0.08 0.08 0.17 0.27 0.51 0.28 0.29 0.21 0.21 0.21 0.21 0.21

Raichle et al. (1976) Fox and Raichle (1986) Seitz and Roland (1992) Fujita et al. (1999) Fujita et al. (1999) Kuwabara et al. (1992) Ginsberg et al. (1988) Ribeiro et al. (1996) Fox et al. (1988) Marrett and Gjedde (1997) Marrett and Gjedde (1997) Vafaee and Gjedde (2000) Vafaee and Gjedde (2000) Vafaee and Gjedde (2000) Roland et al. (1987) Katayama et al. (1986)

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estimates can be made from a meta-analysis of PET results in humans (Table 9.2) that report changes in CMRO2 , CMRglc , and CBF for a wide range of conditions (Figure 9.8(A)). First, there is a minor difference between fractional changes in rates of glucose and oxygen use (i.e., CMRglc /CMRglc and CMRO2 /CMRO2 in Figure 9.8(A)) which suggests that glucose oxidation is still the major source of energy during functional activation in the awake nonanesthetized human brain (Shulman et al., 2001a,b) – similar to the anesthetized rat brain (see above). Second, there is a positive difference between fractional changes in rates of blood ﬂow and oxygen use (i.e., CBF/CBF and CMRO2 /CMRO2 in Figure 9.8(A)) which supports a positive BOLD signal (Equation (9.3)) according to the model of functional hyperemia (Equation (9.2)), even with a signiﬁcant change in oxidative metabolism. The correlation between CBF/CBF and CMRO2 /CMRO2 , which is at the core of the physiological basis of BOLD, for the awake human brain has been a topic of controversy for over a decade because some PET studies (Fox and Raichle, 1986; Fox et al., 1988) had shown that CMRO2 /CMRO2 was negligible in comparison to CBF/CBF or CMRglc /CMRglc during functional activation. The proposal from Raichle and coworkers was that during functional activation the brain’s energy producing pathway switched from an efﬁcient oxidative mode to a less efﬁcient nonoxidative mode. While the small difference between values of CMRglc /CMRglc and CMRO2 /CMRO2 gathered from other laboratories (Figure 9.8(A)) qualitatively supports that CMRglc /CMRglc > CMRO2 /CMRO2 , glucose oxidation still provides more than 90 % of the ATP during functional activation at steady state (Shulman et al., 2001a,b; for details see Rothman et al., 2002). Therefore it is not surprising that the (CMRO2 /CMRO2 )/(CBF/CBF) ratio (∼0.6) derived

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Figure 9.8. Stimulation-induced changes in CMRO2 , CMRglc , and CBF in the awake human brain. (A) The difference between changes in CMRglc and CMRO2 (i.e., CMRglc /CMRglc − CMRO2 /CMRO2 on the vertical axis) reﬂects the degree of glucose oxidation – the lower this value is, the greater the degree of glucose oxidation (see Equation (9.1)). The difference between fractional changes in CBF and CMRO2 (i.e., CBF/CBF − CMRO2 /CMRO2 on the horizontal axis) reﬂects the degree of blood oxygenation – the higher this values is, the greater the BOLD effect (see Equation (9.3)). The pseudo-linear relationship suggests that the BOLD signal changes are commensurate with nearly complete oxidation of glucose during functional activation. Therefore the lack of negligible increase in CMRO2 during functional activation (Fox and Raichle, 1986; Fox et al., 1988) is not in agreement with trends observed in literature. The inset shows the distribution of CMRO2 /CMRO2 , CMRglc /CMRglc , and CBF/CBF values from all the PET studies, where the exact values are in Table 9.2. (B) Comparison of CMRO2 /CMRO2 and CBF/CBF values from PET studies (♦) with calibrated fMRI studies of Hoge et al. (1999) and Kim et al. (1999) shown in gray and black circles, respectively. The data suggest that the (CMRO2 /CMRO2 )/(CBF/CBF) ratio observed in the awake human (∼0.6) is approximately equal to that observed in the rat brain (∼0.7). Exact values of the PET studies are in Table 9.2.

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from majority of the awake human PET and fMRI data (Figure 9.8(B)) is similar to the value (∼0.7) obtained from anesthetized rat data (Figure 9.5(B)). The estimates of the (CMRO2 /CMRO2 )/(CBF/CBF) ratio (∼0.6) from Figure 9.8(B) are in good agreement with more recent PET studies (Mintun et al., 2002). The data shown in Figure 9.8(B) are from the calibrated fMRI studies of Hoge et al. (1999) and Kim et al. (1999) as well as all of the PET studies listed in Table 9.2. As mentioned above, the BOLD calibration experiments that have been conducted until now in humans have used the relationship described by Equation (9.10) to calculate changes in CBV due to the lack of an approved MRI contrast agent that has a very long half-life for human use. But using a value for φ of ∼0.4 in Equation (9.10), instead of ∼0.1 (see above), would underestimate the changes in CMRO2 predicted by the hypercapnia BOLD calibration method (see above). Despite potential underestimation of changes in CMRO2 the relationship between CBF/CBF and CMRO2 /CMRO2 measured by MRI in awake humans is in good agreement with the PET data (Figure 9.8(B)) and the anesthetized rat data (Figure 9.5(B)). However, both the spatial and temporal resolutions are much higher with fMRI. The calibrated fMRI data (Figure 9.6) has spatial and temporal resolutions of ∼0.2 µl and ∼4 s, respectively.

9.9. FUTURE DIRECTIONS In rapidly developing ﬁelds of fMRI, where interests range from physiology to cognition, there is a fascination for measuring the stimulus-induced BOLD signal with very high temporal resolution. This experimental approach is called ‘event-related fMRI’ because the signal is measured following a very short stimulus input (i.e., an event). Although the expectation is that a temporally resolved fMRI signal will reveal new insights into brain function, the time constant of the increase in the BOLD signal cannot be modiﬁed. The slow rate of signal increase is bound by the sluggish mechanical response of the microvasculature. Since each parameter that modiﬁes the BOLD signal (see Equation (9.3)) changes with a distinct time constant, the steady state calibration of fMRI described here cannot be readily extended to interpretation of dynamic neuroenergetic changes from ‘event-related fMRI’ data. Despite the lack of calibration of the various dynamic contributions to the fMRI signal, the ‘event-related fMRI’ data are being interpreted with these as well as other unspeciﬁed assumptions (Buxton et al., 1998; Mandeville et al., 1999; Friston et al., 2000; Zheng et al., 2002). The changes in the fMRI signal can be a delayed positive response (peaks at 4–6 s from stimulation onset; magnitude is high; e.g., Blamire et al., 1992), or an early negative response (peaks at 1–2 s from stimulation onset; magnitude is low; e.g., Menon et al., 1995), or a delayed negative response (peaks at 4–6 s from stimulation onset; magnitude is intermediate; e.g., Harel et al., 2002). While each of these BOLD responses can be explained on the basis of the mechanical manifestations of the microvasculature, only the delayed positive and negative BOLD response, have been calibrated with energetic changes, as shown here. The origins of the early negative BOLD response is still unknown (Logothetis et al., 1999; Buxton, 2001; Shmuel et al., 2002). Despite the lack of statistical reproducibility of event-related fMRI responses and the absence of a causative mechanism, aside from the general assumption that the energetic and hemodynamic effects change independently during the stimulation onset, interpretations of these time courses have taken a central place in the ﬁeld (e.g., Buckner, 2003). Furthermore, the ‘early negative dip’ has been used to obtain columnarlike structures in the visual system (Kim et al., 2000; Duong et al., 2000) despite the unknown origin of the signal, the lack of statistical reproducibility, and the lower sensitivity. In contrast, the superior sensitivity of the delayed BOLD response has demonstrated an overlooked success for mapping columnar-like structures reproducibly in the cerebral cortex of animals (Yang et al., 1996) and humans (Cheng et al., 2001). While many cross-modal correlations (as discussed above) and expectations from psychology and neuroanatomy leave little doubt that the delayed BOLD signal is responding in some way to neuronal activity, the nature of that activity at steady state has now been revealed by calibrated fMRI experiments. Careful

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fMRI calibrations with high temporal resolution are needed before the dynamic fMRI signal can be used to derive values of CMRO2 /CMRO2 , which provide a biophysical relationship between neuroimaging and neuronal activity. The in vivo observations of tight proportionality between CBF and CMRO2 measured regionally at rest and during function, both in the rat (Figure 9.5(B)) and in the human (Figure 9.8(B)), support a concept of the efﬁciency of oxygen transfer from the capillary bed (Hyder et al., 1998, 2000b, 2001). While theories of oxygen transport have not been critical in aiding BOLD calibration at steady state, any future work leading to calibration of the BOLD signal in a time-dependent manner would need to consider distinct and competing steps involved in dynamic oxygen transport from blood to brain. Furthermore this endeavor would require combination of dynamic measurements of S, CBF, CBV, ν, and pO2 in the same model, which is not trivial. However, an even bigger challenge will be to validate the predicted dynamics in CMRO2 . While it is quite possible that the higher temporal resolution of 17 O MRS (Zhu et al., 2002) may play a great role in this process, modifying the POCE method for dynamic changes in CMRO2 is not completely out of the question either. Irrespective of which method is used for the validation process, until dynamic changes in CMRO2 are obtained from calibrated BOLD signals, only steady state calibrated BOLD data provide meaningful neural information.

9.10. CONCLUSION The conventional fMRI map offers information indirectly about localized changes in neural activity since it reliably reﬂects blood oxygenation changes, not alterations in neural activity. To provide neurophysiological basis of fMRI, researchers have combined electrophysiological methods to show correlations of BOLD and electrical signals. But quantitative interpretation of ‘How much has the neural activity changed by?’ still cannot be made from conventional fMRI data. The BOLD signal has two parts, one that describes the energetic support of the bioelectric work (CBF–CMRO2 relationship) to sustain neuronal excitability and the other is the requisite dilatation of vessels (CBF–CBV relationship) presumably involved in removal of waste while providing nutrients. The BOLD signal was calibrated by quantifying these physiological parameters over a wide range of neuronal activity in the anesthetized rat brain. The end result of the BOLD calibration was the ability to calculate changes in CMRO2 . This prediction of CMRO2 changes were also validated with 13 C MRS measurements. Analysis of PET results from the awake human shows that this type of BOLD calibration may provide accurate estimates of changes in CMRO2 with high temporal and spatial resolutions. Pharmacological and electrophysiological studies in the rat revealed that the changes in CMRO2 derived from the BOLD calibration are closely linked with variations in neuronal glutamate release as well as the electrical workload of the neuronal ensemble in a voxel. These ﬁndings conﬁrm the neurophysiologic makeup of BOLD and have taken the necessary steps towards mapping neuronal activity by fMRI.

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10 Relationship between CMRO2 and Neuronal Activity Fahmeed Hyder Department of Diagnostic Radiology and Biomedical Engineering, Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06510, USA

Hal Blumenfeld Department of Neurology and Neurobiology, Yale University School of Medicine, MR Center, P.O. Box 208018, New Haven, CT 06510, USA

10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 10.10 10.11

Introduction Interpretability of Neuroimaging Signals Neuronal Activity and Anatomy Measuring Changes in CMRO2 and ν Relationship between CMRO2 and ν Comparison with Previous Studies Redeployment of Spiking Frequency Distributions Implications for Interpretation of fMRI Biophysical Basis of Cognitive Neuroscience Future Directions Conclusion

Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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10.1. INTRODUCTION Neuroimaging methods highlight speciﬁc areas of the human brain which are involved in the sensation, perception, and cognition of stimuli. Following the introduction of PET for brain mapping about two decades ago (Raichle, 1988), fMRI has come forward to play the more dominant role in both human and animal functional studies of the brain (Shulman, 2001). A modern graduate student of neuroscience would certainly have seen remarkable colored PET or fMRI images of the working human brain in text books. And why not? Both PET and fMRI methods, the former being a little more invasive than the latter, are at the forefront of cognitive and clinical neuroscience research today. The typical student’s interpretation of the superb colored hues representing the activated spots would probably be that speciﬁc brain regions work when they are provoked by the stimulus. This appraisal of the active regions identiﬁes those that survive the differencing away of the baseline, i.e., when the subject is doing ‘nothing’ but resting inside the scanner (Posner and Raichle, 1998). This interpretation is based on the cognitive neuroscientists’ belief that doing ‘nothing’ during the baseline condition actually meant that the brain cells were not doing anything that was meaningful for the task in question (Shulman and Rothman, 1998). However, the results to be discussed in this chapter show that the baseline neuronal activity contains, for neuroscience and interpreting functional imaging, the most important information. The functional integrity of the working brain is maintained by communication amongst an enormous number of neurons (Figure 10.1(A)). While neurons transmit and receive electrical signals across synapses (Kandel et al., 1991), the participation of astrocytes (becoming recognized as the neurochemical collaborator in cellular activity (Pellerin and Magistretti, 1994)) in neurotransmission is an integral part of synaptic communication (Figure 10.1(B)). The communication between neurons is characterized by an electric discharge – otherwise known as an action potential (or a spike) – which is followed by a quiescent charging period (Figure 10.1(C)). At rest the cell membrane is nearly impermeable to Na+ ions, which have a greater concentration outside than inside the cell. However, during an action potential Na+ conductance rapidly increases and Na+ ions move into the cell, making the voltage inside the cell (Vm ) more positive, approaching the Na+ ion equilibrium potential (ENa ). The rapid change in Vm triggers a delayed increase in K+ conductance (gK ) as well as a delayed decrease in Na+ conductance (gNa ). These conductance changes combine to cause the Vm to ultimately move back towards the resting value. Thus the action potential has a very short life time (1–2 ms), characterized by a fast depolarization (Na+ inﬂux) phase followed by an equally rapid hyperpolarization (K+ efﬂux) segment (Hodgkin and Huxley, 1952). Action potential generation is driven by gradients of Na+ and K+ ions across the cell membrane. Much like batteries powering an electric device, these gradients (high Na+ outside the cell and high K+ inside the cell) must be maintained for repeated spikes to occur. Active Na+ –K+ pumps consume large amounts of ATP to maintain these ionic gradients so that the neurons are ready to work (i.e., ‘ﬁre’) whenever called for (Ritchie, 1967). The discharging and charging phases are traditionally referred to as ‘ﬁring’ and ‘nonﬁring’ periods (Figure 10.1(D)). However, these epochs are not exactly binary because cations are continuously moving across the cell membrane, either via passive diffusion or evoked by a signal from a pre-synaptic neuron, causing Vm to ﬂuctuate. Since an action potential is generated only if Vm rises beyond a speciﬁc threshold in the post-synaptic neuron, the intermittently changing Vm creates waves of synaptic ﬁeld potentials that have a longer lifetime (10–20 ms) as a consequence of failed attempts of the post-synaptic neuron at creating spikes. Thus the main difference between measures of action and ﬁeld potentials is that the former is readily quantiﬁable because a speciﬁc rate of ﬁring can be assigned for individual neurons, which allows for discrimination between spontaneous ﬁring and modiﬁed intracellular potentials due to incoming signals (Gray, 1995).

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Figure 10.1. Neuronal activity and energetics. (A) Neurons communicate via synapses and are surrounded by astrocytes (shown in gray). (B) The electrical and chemical synapse at a magniﬁed scale showing pre- and post-synaptic elements, along with astrocytes and capillaries. Action potentials in the pre-synaptic neuron (shown on the right inset) trigger neurotransmitter release, which in turn excite the post-synaptic neuron. This results in post-synaptic potentials which can be recorded from the post-synaptic neuron or from the extracellular space as ﬁeld potentials (as shown on the right inset). Speciﬁc channels for cations allow permeability of Na+ and K+ ions across the cell membrane during action potentials. Na+ –K+ pumps help maintain the normal ionic gradients across the cell membrane. Since ATP is consumed in neurons (and astrocytes) for maintenance of the ionic gradients, the energy pool is replenished by substrate delivery from capillaries. (C) Simulated changes in cell membrane voltage (Vm ), sodium conductance (gNa ) and potassium conductance (gK ) in a neuron during an action potential (Hodgkin and Huxley, 1952). At rest, Vm is close to the potassium equilibrium potential (EK ), while at the peak of the action potential Vm is close to the sodium equilibrium potential (ENa ). (D) A small portion of an extracellular recording demonstrating repeated ﬁring of a single neuron from the α-chloralose-anesthetized rat brain. See Smith et al. (2002) for details.

10.2. INTERPRETABILITY OF NEUROIMAGING SIGNALS An ideal way to probe the activity of neurons would be to measure electrical signals directly with tiny electrodes. However, simultaneous measurement of a large number of neurons is limited by the number

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of electrodes that can practically be implanted and recorded from the intact brain. While neuroimaging methods are obviously more practical for noninvasive experiments in humans, the techniques to localize neuronal processes are based on the assumption that local activity of a neuronal ensemble changes when a region is responding to a task. Electrical activity in the brain has signiﬁcant associated energetic costs which are mainly related to ion movements across axonal and synaptic membranes (Ames, 2000). Since neuronal work requires oxidative energy and oxygen reserves in the brain are extremely low (Siesjo, 1978), supplies of nutrients (i.e., glucose and oxygen) are tightly regulated by the high energy demands of neuronal work (Figure 10.2). Thus neuronal activity is inferred from measurements of cerebral blood ﬂow (CBF) as well as cerebral metabolic rates of glucose (CMRglc ) and oxygen (CMRO2 ) utilization by neuroimaging methods. Usually PET and fMRI are applied in a relative sense where the fractional signal changes in speciﬁc brain regions reveal where the neuronal activity has changed. But the degree by which neuronal activity has changed cannot be made from the differencing approach because the discard of the ‘baseline’ signal assumes the noncontributing nature of this signal for sensation, perception, and cognition. Given the widespread use of the differencing approach in cognitive neuroscience (Shulman, 1996), one might expect concerns about how the spontaneous baseline activities are related to the colored hues in the functional images. Surprisingly, however, this is far from the case. To connect the neuroimaging signal to neuronal activity requires establishing two relationships: (i) associating the neuroimaging signal to localized neuroenergetics; (ii) establishing the connection between energy consumption and neuronal processes related to information transfer (e.g., rates of neurotransmitter release and/or action potential propagation). Recent calibration studies of fMRI in rat brain have established the ﬁrst relationship (see Chapter 9), where the fMRI signal changes (S) were correlated with alterations in energy use (CMRO2 ) during glutamatergic neurotransmission. Some years ago Sokoloff and coworkers explored the second relationship in peripheral neurons with 2-14 C-deoxyglucose autoradiography and electrophysiology (see Chapter 2) to show that energy consumption (CMRglc ) associated with electrical activity is roughly proportional to spiking frequency (ν). Recent 13 C MRS studies of anesthetized rat brain explored the 8.0

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Figure 10.2. Relationship between CBF and CMRO2 . The near-linear relationship between CBF and CMRO2 , as measured by NMR (•) and non-NMR () methods from isoelectric conditions to awake cortical activity, allows the fMRI signal changes to be quantitatively used for neuroimaging (for a recent review see Hyder et al., 2001). The scale on the right inset shows qualitatively the levels of cerebral activity, ranging from isoelectric conditions to awake cortical activity, where the nonanesthetized resting awake level is marked by the dotted lines. The details of the different CBF and CMRO2 measurements are in Chapter 9.

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second relationship further (see Chapter 5), where a stoichiometric relationship was measured between rates of neuronal energy metabolism (CMRO2 ) and neurotransmitter cycling (Vcyc ). Although these combined results suggest that CMRO2 is related to ν and Vcyc in glutamatergic synapses, technical limitations of studying the energetics of peripheral neurons or a heterogeneous group of cerebral neurons has limited the quantitative interpretability of these ﬁndings for neuroimaging, specially for fMRI. In this chapter the second relationship is explored further by examining the dependence of spiking frequency (ν) of a neuronal ensemble, primarily consisting of pyramidal neurons, on localized energy metabolism (CMRO2 ) in the anesthetized rat brain. The evidence to be presented shows a positive and close to linear relationship between the functional increments in energy consumption (CMRO2 ) and the average rate of neuronal ﬁring (ν), which suggests that regional brain energy metabolism provides the most direct correlate of spiking activity of a neuronal ensemble. Furthermore the electrophysiological data of the neuronal ensemble and the distribution of spiking frequencies show that the differences induced by stimuli require deployment of most of the neurons in the nonstimulated baseline state, thereby emphasizing the importance of the total or summed activity of the ensemble.

10.3. NEURONAL ACTIVITY AND ANATOMY Neuronal communication depends on electrical activity in the form of dynamic changes of Vm in massive populations of excitatory and inhibitory neurons. The changes in Vm allow signals to propagate within single neurons and to subsequently be communicated between neurons across the chemical and electrical synapses (Figures 10.1(A) and (B)). A variety of methods are available for studying the electrical signals from neurons in vivo. The spatial scale of the measurements range from membrane potentials of a single neuron to macroscopic potentials recorded from large regions (for a review see Kandel et al., 1991). What is the most appropriate electrical measurement for determining neuronal ensemble activity in relation to energetics? The methods for single cell recordings involve obtaining direct access into the intracellular milieu so that Vm can be measured with high impedance electrodes, which consist of ﬁne glass capillary tubes ﬁlled with electrolytes. The disadvantage of intracellular recordings in studying populations of neurons is the difﬁculty of sampling large number of neurons because only one neuron can be studied with each electrode. An alternative to this approach is extracellular recordings with glass capillary or metallic electrode tips placed proximally to cell bodies so that the extracellular voltage can be measured. The action potentials from a single neuron can be resolved by adjusting the position of the electrode tip (i.e., single-unit activity (SUA) recording). An electrode with slightly lower impedance can detect action potentials from more neurons with a single penetration (i.e., multi-unit activity (MUA) recording). The neurons closest to the electrode tip generate the largest signals and these signals can be separated by spike sorting algorithms. The unresolved signals from low impedance extracellular electrodes also contain the slow waves of synaptic ﬁeld potentials. The advantage of extracellular recording is that several individual neurons can be identiﬁed and therefore studied simultaneously. Other methods such as magnetoencephalography (MEG), electroencephalography (EEG), and somatosensory evoked potentials (SEP) detect summed post-synaptic potentials from cells located both near to and far from the recording elements, which are placed on the scalp and have very low impedance. These methods are less useful for quantitative measurements because the signals lack spatial speciﬁcity. Some uncertainties about the lack of spatial speciﬁcity of EEG and MEG can be diminished by using slightly higher impedance wires placed in the brain to measure local ﬁeld potentials (LFP). However the LFP signals are not easily quantiﬁable because these signals integrate over space and time and do not have the all-or-none speciﬁcity of spikes. Thus, at the present time, the best method for measuring the activity of a population of neurons in vivo is extracellular unit recording, because the data can be quantiﬁed in terms of spiking rates of an ensemble of neurons and sampled continuously for variety of conditions.

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Figure 10.3. Cellular layers of the rat somatosensory cortex. (A) The cortical layers in gray matter are numbered 1–6 from the pial surface moving inward towards the white matter. (B) A magniﬁed view of the cortical layers in the rat somatosensory region. The layers can be distinguished microscopically by their content of different cell density and sizes (see left inset). The small network connections across different layers vary: layer 1 composed mainly of axons and dendrites, layers 2 and 3 of small to medium pyramidal neurons with major cortical–cortical connections, layer 4 of densely packed pyramidal and nonpyramidal neurons receiving inputs from the thalamus, layer 5 of large pyramidal neurons which project to the brainstem and spinal cord, and layer 6 of modiﬁed pyramidal cells with major reciprocal projections to the thalamus.

The majority of the forebrain surface in most mammals consists of a six-layered neocortex (Figure 10.3(A)). Most neocortical regions contain laminae, which are numbered 1 through 6, progressing from the pial surface to the white matter. Layer 1, or the molecular layer, is sparsely cellular and contains mainly axons and apical dendrites of neurons from other regions. Layers 2 and 3 contain mainly small and medium sized pyramidal neurons, respectively. Layer 4, or the granular layer, consists of small densely packed pyramidal and nonpyramidal neurons. Layer 5 contains medium and large pyramidal neurons. Layer 6 consists mainly of the modiﬁed spindle-shaped pyramidal neurons. Extensive synaptic connections occur in the cortex in the vertical (radial) direction (Lorente de No, 1938), giving rise to functional modules or columns. In sensory systems, individual neurons oriented vertically are frequently arranged into functional units, e.g., in the cerebral cortex columns or barrels to recognize speciﬁc characteristics of stimuli (Woolsey, 1996; Mountcastle, 1997). However, horizontal connections are also prominent, and many of these are collected in plexuses of axon ﬁbers traveling in layer 1, in layer 4, and in the deep portions of layers 5 and 6 (Nieuwenhuys, 1998). The major fraction of neocortical neurons (∼80 %) consists of pyramidal neurons (Winﬁeld et al., 1980; Peters et al., 1985) (Figure 10.3(B)). Their cell bodies have a pointed top, oriented towards the surface of the neocortex giving rise to a large apical dendrite, and a relatively ﬂat bottom giving rise to a skirt of basal dendrites. The pyramidal cell axon emerges from the base of the cell and typically gives rise to extensively ramifying collateral branches which reach other cortical neurons as well as subcortical structures. The pyramidal neurons are excitatory and utilize glutamate as the neurotransmitter. Other nonpyramidal neurons (e.g., basket cells, chandelier cells, etc.) make up the

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remainder of the neocortical neuronal population. Most nonpyramidal neurons form local cortical circuits and are inhibitory utilizing γ -aminobutyric acid (GABA) as the neurotransmitter. In addition, there are some excitatory nonpyramidal neurons which also form local cortical circuits (e.g., spiny stellate cells, bipolar cells, etc.) and are primarily found in the primary sensory areas. The largest number of synapses in the neocortex is formed by pyramidal neurons (Thomson and Deuchars, 1997). Although the cortex has reciprocal input and output connections with subcortical structures (e.g., thalamus), the fraction of cortico-cortical connections are far greater (∼85 % of synapses are corticocortical). Axon collaterals of pyramidal neurons form a vast network interconnecting pyramidal neurons via short-range connections. Nonpyramidal local circuit neurons also contribute to the complex network of cortico-cortical connections. In addition, projecting axons from pyramidal neurons form long-range connections between cortical regions both within and across cerebral hemispheres. Although cortico-cortical connections occur between all cortical layers, they are most prominent between pyramidal neurons in the supragranular layers 2 and 3 and to a lesser extent in layer 5 (Gottlieb and Keller, 1997). Despite this massive cortical intraconnectivity, inputs from subcortical structures appear to serve as crucial ‘drivers’ for the information processed by many cortical regions. Information processing in primary sensory areas depends heavily on visual, auditory, or somatosensory inputs relayed to the cortex via the thalamus (∼10 % of synapses are thalamo-cortical). The thalamus relays input to the cortex from many subcortical sources including motor information from the basal ganglia and cerebellum, limbic pathways, arousal systems, and other higher-order processing systems. The thalamus, in turn, receives major reciprocal projections from the cortex. Various classiﬁcations have been described for the laminar distribution of thalamic inputs into nearly all cortical layers (Lorente de No, 1938; Macchi, 1983; Herkenham, 1986; Castro-Alamancos and Connors, 1997). However, the most prominent of these projections for primary somatosensory, visual, and auditory processing reach cortical layer 4 and lower parts of layer 3. In addition to thalamic inputs other sources of subcortical projections to the cortex include cholinergic and GABAergic inputs from the basal forebrain, histaminergic inputs from the posterior hypothalamus, dopaminergic inputs from the ventral tegmental area, serotonergic inputs from the raphe nuclei, noradrenergic inputs from the locus ceruleus, as well as various peptides and other modulators. In summary, cortical layer 1 contains mainly axons and dendrites arising from other layers, layers 2 and 3 contain pyramidal cells projecting mainly to other cortical areas, layer 4 receives dense sensory inputs from speciﬁc thalamic relay nuclei, layer 5 projects to the spinal cord, brainstem and basal ganglia, and layer 6 projects in a reciprocal manner to thalamic relay nuclei. Intracortical connections predominate, but cortical inputs and outputs play a crucial role in signal processing. Cortical regions demonstrate different laminar organization based on their functions. Thus primary sensory regions such as the visual, auditory, or somatosensory cortex have a very prominent layer 4, which receives the majority of the sensory input from the thalamus, and a relatively thin layer 5. Primary motor cortex has a thick layer 5, and a sparse layer 4. Association cortex is intermediate in laminar organization between these two extremes.

10.4. MEASURING CHANGES IN CMRO2 AND ν As stated earlier, this chapter focuses on our recent studies where quantitative changes in CMRO2 and ν were measured (Smith et al., 2002; Hyder et al., 2002). All methods described below were applied in αchloralose-anesthetized rats (Sprague-Dawley; male) and systemic physiologic parameters were monitored and controlled. In these rats the halothane exposure during surgery was minimized (<1 h). Since halothane has a slow washout (Venkatasubramanian et al., 1996), it is important to exclude the lingering effects of halothane from α-chloralose (Ueki et al., 1992). The same forepaw stimulation (2 mA; 0.3 ms duration, 3 Hz) was applied at high (condition I, light anesthesia) and low (condition II, deep anesthesia) baseline levels where the depth of anesthesia was set by α-chloralose dosage. The anesthesia dosages for the high

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(condition I, light anesthesia) and low (condition II, deep anesthesia) baseline levels were determined from the total amount of α-chloralose given (i.e., bolus + supplemental doses) over the total time expired during measurement. The light and deep anesthesia levels (i.e., conditions I and II) were achieved with α-chloralose dosages of 36 ± 2 and 46 ± 4 mg/kg/h, respectively. A 2–5 min stimulation protocol in a block design was repeated a few times (in each baseline condition) with ∼10 min resting periods. After a stabilization period of ∼1 h in between light and deep anesthesia levels (i.e., conditions I and II), the data for the deep anesthesia level (i.e., condition II) were collected in a similar manner as in the prior light anesthesia level (i.e., condition I) in the same rats. The calibrated fMRI experiments, conducted on a 7 T horizontal bore system, were used to calculate CMRO2 /CMRO2 (see Chapter 9). Brieﬂy the method is described as follows. At steady state the fMRI signal change ( S/S) is related to the metabolically driven increase in perfusion. Since fMRI has been calibrated over a certain dynamic range for the rat brain (see Chapter 9), multimodal measurements of changes in CBF ( CBF/CBF) and the fMRI signal ( S/S) allow changes in CMRO2 (CMRO2 /CMRO2 ) to be calculated CMRO2 CBF S −1 = (10.1) − M +ρ CMRO2 CBF S where M is measurable physiologic and magnetic constant and ρ is a small measurable component that is related to changes in blood volume (see Chapter 9). The relationship measured between CMRO2 and CBF (Figure 10.2) shows that CMRO2 /CMRO2 is approximately equal to CBF/CBF in the rat brain. More importantly the changes are linear over small ranges in activity. The changes in CBF generally exceed changes in CMRO2 for S/S > 0 (i.e., functional hyperemia, as described in Chapter 9). For both light and deep anesthesia levels (i.e., conditions I and II), activation foci (of CMRO2 /CMRO2 ) were obtained by using the same t-statistical threshold. A localized region of 2 × 2 voxels in the somatosensory forepaw region (layer 4) was interrogated for both stimulations from light and deep anesthesia levels (i.e., conditions I and II). All electrophysiologic studies were guided by the localization of prior fMRI results (within accuracy of ∼100 µm). The rat was placed prone on a stereotaxic holder. The skull above the contralateral somatosensory forepaw region (4.4 mm lateral and 1.0 mm anterior to bregma) was opened via a small burr hole and high impedance (2–4 M) tungsten–iridium microelectrodes (tip <1 µm) were inserted to a depth of cortical layer 4 (Figure 10.3) using stereotaxic manipulators. Prior to digitization, the raw extracellular signals were ampliﬁed and ﬁltered. Spiking activity of the neuronal ensemble was extracted from extracellular data by shape recognition of high signal-to-noise ratio (SNR) spikes using typical commercial software. Other details are in Smith et al. (2002). For simplicity in describing the spike analysis procedure as well as the results, the light and deep anesthesia levels are referred to as conditions I and II, respectively, from here on. For data obtained in the baseline period of condition I (light anesthesia), spike analysis was used to create a template of single units, with a maximum of two neurons per electrode (Figure 10.4(A)). This approach allowed identiﬁcation of some electrical stimulation artifacts (due to inadequate grounding in some rats) in the extracellular data (Figure 10.4(A)), which could then be easily discarded from the spiking activity data. The artifactfree template was then used to extract the spiking activity from the extracellular data (Figure 10.4(B)) such that a temporal history for each experimental run could be created. The spiking data were then converted to reﬂect the relative spiking frequency of the ensemble (ν) by analyzing consecutive 10 s bins (Figure 10.4(C)). The template created in condition I (light anesthesia) was also used to process the data collected in condition II (deep anesthesia). In all cases an additional template was created from the data in condition II. If the templates in conditions I and II (from the baseline periods only) did not match by the criteria of amplitude (±20 %) and shape (±0.1 ms), the data set from that rat was discarded. This approach assured, by the standards speciﬁed above, that the spiking data extracted from extracellular data were from the same neuronal ensemble in both conditions (per rat). Since ν was measured in both conditions for

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Figure 10.4. Measuring neuronal ﬁring in the rat somatosensory cortex. (A) Analysis of extracellular data with commercial spike sorting software (e.g., see Zouridakis and Tam, 2000) was used to create a template of single units. This approach usually provided the recognition of two spike shapes with high SNR per electrode (top trace; spikes 01 and 02) and in some cases allowed the recognition of the stimulus artifact (bottom trace; spike 03). The time window in all spike shapes shown was 1.6 ms. The top and bottom templates were obtained from different rats. (B) The created template was then used to create a temporal history of spiking activity (top trace) extracted from the raw extracellular signal (bottom trace). (C) The spiking activity was then examined in consecutive 10 s bins to reﬂect the relative spiking frequency of the neuronal ensemble (ν) over the entire experimental run. The thick bar represents the forepaw stimulation period. All data are from rats in condition II. See Smith et al. (2002) and Hyder et al. (2002) for other details.

each rat, the changes in spiking frequency (ν/ν) was used to reﬂect the behavior of the ensemble for all conditions.

10.5. RELATIONSHIP BETWEEN CMRO2 AND ν The stimulation-induced changes in spiking frequency of an ensemble of neurons (ν) were compared to changes in energy use (CMRO2 ) for two different depths of α-chloralose anesthesia (i.e., conditions I and II for light and deep anesthesia, respectively). Figure 10.5 shows the localized changes in CMRO2 and ν from the contralateral cortex during forepaw stimulation, whereas no signiﬁcant changes were observed in the ipsilateral side (data not shown). In each baseline condition the same stimulation was applied, resulting in a total of four activity levels consisting of two baseline and two stimulated conditions. Thus there were three transitions: stimulation-induced increment from baseline of condition I, anesthesia-induced decrement from baseline of conditions I to baseline of condition II, and stimulation-induced increment from baseline of condition II. In the same rat, the same forepaw stimulation induced different increments in CMRO2 (by ∼40 % and ∼100 %) from the baselines of conditions I and II (Figure 10.6). The same stimulation increased the contralateral values of ν by similar magnitudes (∼30 % and ∼100 %), in good agreement with the changes in CMRO2 (Figure 10.6). There was also a transition between baselines of conditions I and II, a decrement of ∼30 % in both CMRO2 and ν (Figure 10.6). Increments in both CMRO2 and ν were greater

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Figure 10.5. Localized changes in energy metabolism (CMRO2 ) and spiking frequency (ν) of a neuronal ensemble. In each case, the fMRI and electrophysiology measurements were made in each rat ﬁrst under high basal activity (condition I; left) then under low basal activity (condition II; right), using two different doses of α-chloralose. The CMRO2 /CMRO2 maps (overlaid on stereotaxic coordinates from Paxinos and Watson, 1997) were determined from calibrated fMRI (see Chapter 9), whereas the ν/ν values were extracted from extracellular recordings (see Figure 10.4). Approximately 92 % of all microelectrode penetrations (in layer 4) identiﬁed at least two discrete neurons from the spike analysis (see Figure 10.4). No signiﬁcant stimulation-induced changes in ν and CMRO2 were observed in the ipsilateral side (data not shown). All data adapted from Smith et al. (2002) and Hyder et al. (2002).

from baseline of condition II than from baseline of condition I. These measured changes in CMRO2 and ν compare well with prior studies from other laboratories. First, the localized signals detected in these anesthetized studies should not be an issue of controversy because it is accepted that the stimulationinduced increments in somatosensory cortical activity (e.g., as measured by changes in CMRglc and CBF) in anesthetized animals provide localization of columnar units which agree with spatial activation patterns in nonanesthetized animals (Sokoloff, 1991). Second, it is well known that both neuronal activity and energetics are decreased with various anesthetics (e.g., halothane, α-chloralose, phenobarbital) in a dosedependent manner (Savaki et al., 1981; Dudley et al., 1982; Hodes et al., 1985). Third, magnitudes of stimulation-induced increments are anesthesia dependent (Harding et al., 1979; Bonvento et al., 1994; Shulman et al., 1999). When each stimulation-induced increment (in both CMRO2 and ν) from conditions I and II (i.e., light and deep anesthesia levels) was added to the respective baseline value, approximately similar total values for conditions I and II were reached (Figure 10.7(A) and (B)). The data shown in Figure 10.7(C) were normalized to the nonstimulated (values of CMRO2 and ν in) baseline of condition I. The increments of CMRO2 and ν upon forepaw stimulation elevated each signal to the same ﬁnal values in both conditions, in agreement with previous suggestions from the literature (for a review see Shulman et al., 1999). The current results show that ν/ν is approximately equal to CMRO2 /CMRO2 , which is in agreement with

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Figure 10.6. Relationship between energy use ( CMRO2 /CMRO2 ) and spiking frequency (ν/ν) of a neuronal ensemble. All rats were anesthetized with α-chloralose, where the baseline was changed by the dosage level. The ﬁlled bars represent stimulation-induced incremental changes in CMRO2 and ν from baseline of condition I (light gray) and baseline of condition II (dark gray) in the same rats for each method. The open bars represent the decrement of baseline from condition I to II. The incremental change upon stimulation from baseline of condition II was larger. The CMRO2 /CMRO2 values were obtained from a total of 24 voxels (from all rats; n = 6) placed over the forelimb region (other activated voxels were not analyzed). The ν/ν values were obtained from extracellular recordings of a total of 72 neurons in layer 4 of the forelimb region (from all rats; n = 36). The ∼1:1 relation between CMRO2 /CMRO2 and ν/ν agrees with results from Chapter 5. All data adapted from Smith et al. (2002) and Hyder et al. (2002). 0.8

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Figure 10.7. Absolute changes in changes in energy metabolism (CMRO2 ) and spiking frequency (ν) of a neuronal ensemble. The temporal changes in (A) CMRO2 and (B) ν are shown relative to the baseline of condition I, where the stimulation duration is shown by the vertical lines. (C) While the increments (ﬁlled) of CMRO2 and ν were different in conditions I (gray) and II (black), the same total activity levels were reached upon stimulation, where the size of the increment was dependent on the baseline level (open). See Figure 10.6 for support and signiﬁcance because the incremental changes (ﬁlled) can be directly compared. All data adapted from Smith et al. (2002) and Hyder et al. (2002).

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linear relationships between CMRglc and ν in peripheral neurons (see Chapter 2) and CMRO2 and Vcyc in cortical neurons (see Chapter 5). Therefore the combined results suggest Vcyc CMRO2 ν ≈ ≈ CMRO2 Vcyc ν

(10.2)

which means that both electrical (ν) and chemical (Vcyc ) activities of glutamatergic neurotransmission are proportional to the oxidative energy consumed (CMRO2 ). Thus neurotransmission (i.e., work done) is directly coupled to neuroenergetics (i.e., energy consumed). Because the term ‘work’ is deﬁned here as the sum of the energy consuming events associated with neuronal activity, energy connects electrical and chemical aspects of neural work (Shulman et al., 2002).

10.6. COMPARISON WITH PREVIOUS STUDIES An unavoidable and obvious outcome of this type of multimodal experiment with fMRI and electrophysiology is that there are consequences for the neurophysiologic interpretation of fMRI. The correlations that have been observed between fMRI and electrical signals from neurons are based on features of either matching temporal dynamics or generating spatial overlaps (Yang et al., 1997; Brinker et al., 1999; Ogawa et al., 2000; Rees et al., 2000; Logothetis et al., 2001). Despite large disparities in temporal dynamics as well as the inherent ambiguities about spatial resolution of the signals, these correlations only conﬁrm that one event is in spatial proximity with the other. Since each signal depends upon varying dynamics, any conclusion beyond the spatial anchoring of the two signals would lack biophysical foundations. The ease of ﬁnding temporal and/or spatial correlates, depending upon vague properties, in part explains why a quantitative relationship between fMRI and electrical signals (as measured by EEG, SEP, LFP, SUA, MUA, and ν) has been rather controversial (see below). These correlations, however, do not account for the grouped activity of an ensemble of neurons in the voxel. Why? Because an fMRI signal even from a small voxel of 0.001 µl, which is the highest resolution achieved (see Chapter 9), contains thousands of cortical neurons. Despite these limitations of phenomenological correlations, we discuss a few studies in detail because the results which have apparently been accepted, despite the absence of convincing mechanisms, are having major impacts on the neurophysiologic basis and interpretation of fMRI. Logothetis et al. (2001), working on the primary visual cortex of anesthetized primates, concluded that changes in the fMRI signal represent neuronal input because they observed a higher correlation between time courses of the fMRI signal (a response delayed response by several seconds following the stimulus onset) and the LFP signal (an almost instantaneous response following the stimulus onset). The LFP signals are believed to reﬂect mainly the neuronal input because they represent the weighted sum of changing membrane potentials in the dendritic branches and the soma as a consequence of spiking from neighboring neurons. Since input and output signals are not entirely distinct in a neuronal ensemble especially in layers 4 and 5 (i.e., site of the electrical measurements) where the nonpyramidal neurons form numerous cortico-cortical synapses (Figure 10.3), it seems premature to link the fMRI signal to either the input or output of the system simply due to a better temporal correlation. Furthermore the slightly worse correlation between the time courses of SUA (or MUA) and fMRI signals could in part be attributed to the higher SNR of LFP signals. Rees et al. (2000) tried to relate the stimulation-induced increment in the fMRI signal in humans with changes in ν measured in non-human primates. Despite concerns about cross-species comparison of signals from two entirely different modalities, they suggested that a ∼1 % change in the fMRI signal (at 1.5 T in humans) corresponds to ∼9 Hz change in the ν per neuron in region V5 of the non-human primate visual cortex. Thus they concluded that the fMRI signal magnitude is better correlated with the spiking activity (i.e., the neuronal output), in direct contrast to the conclusion of Logothetis and colleagues. Similarly other studies have shown that the magnitude of the fMRI signal change correlates

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well with SEP (Brinker et al., 1999) and the evoked EEG (Ogawa et al., 2000) signals. Since the origin of different electrical signals measured is quite varied, which correlation between the fMRI signal and neuronal activity is to be trusted? However, Ogawa et al. (2000) report an approach by which ‘some type of’ stimulation-induced neuronal events can be probed by fMRI signal with temporal resolution in scale of tens of ms. A dual-stimulation paradigm (e.g., bilateral forepaw stimulation separated by a delay), which generates distinct localized activations, shows that the response due to the ﬁrst (contralateral forepaw) stimulation as measured by EEG or fMRI is affected by the delayed second (ipsilateral forepaw) stimulation. While a ‘refractory’ period of ∼40 ms was measured for the rat forepaw stimulation model, in the human visual cortex the measured ‘refractory’ period was ∼100 ms. Despite the unknown origins of this type of interaction from two neuronal events occurring at the same cortical site, this approach has received some attention perhaps due to the fact that ‘by controlling the temporal relation of input tasks it is possible to study temporal evolution of certain neural events at the time scale of their evoked electrical activity by noninvasive fMRI methodology.’ Inter-hemispheric connections (McKenna et al., 1981) may in part explain this type of interaction and interactions from these dual-stimulation paradigms, which depend upon delays that reﬂect complex brain activity, may provide some insight into the neurophysiological basis of fMRI. The term ‘neuronal activity’ has been applied to a host of energy requiring processes including action potential generation and propagation, maintenance of resting potentials, neurotransmitter release and uptake, vesicular recycling, and pre-synaptic Ca2+ currents (see Chapter 7). All of these processes are involved in short- and long-term signal encoding. In glutamatergic synapses of the mammalian brain, an ‘excited’ neuron generates short-lived (1–2 ms) action potentials which propagate (scale of µm) down the axon to initiate Ca2+ -triggered glutamate neurotransmitter release via exocytosis of vesicles from the pre-synaptic neuron. The release of glutamate into the extracellular space produces dendritic ﬁeld potentials in nearby post-synaptic neurons that can integrate over time (10–20 ms) and space (scale of mm) to generate new trains of spikes. The astrocytic uptake of glutamate and its recycling to the pre-synaptic neuron is another critical energy consuming step in neurotransmission. These processes in glutamatergic neurons, as well as associated ion conductivity in glia and GABAergic neurons, all require energy because active Na+ –K+ pumps utilize ATP to restore Na+ and K+ concentrations across the cell membrane (Figure 10.1(B)). Do brain cells utilize energy by an amount equal to the degree of work done? If so, then energy can be deﬁned as the capability of doing work – a fundamental biophysical principle (Garby and Larsen, 1995). In our study spiking frequency of a neuronal ensemble (ν) was used as a reliable and quantiﬁable measure of brain work. Likewise, calibrated fMRI was used to measure changes in energy utilization (CMRO2 ). The results showed that change in energy utilization (CMRO2 /CMRO2 ) was approximately equal to change in work done by the neuronal ensemble (ν/ν). The relationship found in this study between changes in CMRO2 and ν (Figures 10.5–10.7) has the advantage over previous studies of not depending upon complex electrophysiological (e.g., electrode impedance, ﬁltering process) or fMRI (e.g., ﬁeld strength, pulse sequence) parameters. These issues were effectively normalized by the fMRI calibration and validation (see Chapter 9), detecting the same neuronal ensemble for the entire experiment (in the same rat), and by using the same stimulation for both baseline conditions (in the same rat). Hence the fMRI signal change (i.e., S/S in Equation (10.1)) when calibrated to provide CMRO2 /CMRO2 , allows a quantitative relationship between energy consumption and the rate of spiking activity (Figure 10.6). This offers a biophysical relation between the neuroimaging signal and a basic measurement of neuronal activity, which agrees with the relationship between CMRO2 and Vcyc obtained from 13 C MRS studies (Equation (10.2)). The apparent paradox of a range of energy consuming processes being proportional to a single electrical activity may be resolved if these processes are all coupled to the averaged rate of the electrical activity, which in this case is the ﬁring of an ensemble of pyramidal neurons. This coupling has been proposed by

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Laughlin (2001) who calculated the distribution of energy amongst the different processes (see Chapter 7). The results suggest that most of the energy associated with signaling in the mammalian cortex is coupled to the ensemble ﬁring rate of pyramidal neurons. The calculations show that at a resting spiking rate of 4 Hz (per neuron) more than 80 % of the total ATP is utilized for functional processes coupled to glutamate release, most of which is used for restoring ion gradients. While the efﬁcient use of energy at rest is in good agreement with previous 13 C MRS measurements (see Chapter 5), the proportionality between energy use and work done (i.e., Equation (10.2)) supports the view that the brain spends most of its resources to reestablish ionic gradients so that neurons are ready to ﬁre whenever necessary. These energy budget estimations by Laughlin (2001), relying on morphologic and functional data of rat brain, stand in contrast to previous estimates by Creutzfeldt (1975) which were based on data obtained from the giant squid axon inappropriately extended to the mammalian brain.

10.7. REDEPLOYMENT OF SPIKING FREQUENCY DISTRIBUTIONS In our study, values of ν/ν were determined from extracellular recordings and compared with CMRO2 / CMRO2 from calibrated fMRI of the same voxels (Figures 10.5–10.7). Predominantly pyramidal neurons were measured in each rat. Since more than 90 % of all microelectrode penetrations yielded at least two discrete and recognizable neurons, multiple electrode measurements provided a reasonable activity estimate of the entire neuronal ensemble (n = 72). The histograms of ν for all four conditions (Figure 10.8(A) and (B)) show that during stimulation the distributions became similar. Statistically signiﬁcant increases and decreases in ν were observed upon stimulation in ∼60 % and ∼10 % of the recordings (in condition II), respectively, whereas in the remaining population no signiﬁcant changes were observed (Figure 10.8(C)). Although similar behaviors of neuronal ensembles in the cortex have been reported previously (McCasland and Woolsey, 1988; Scannell and Young, 1999), the quantitative redeployment of the same ensemble for different sensory workloads (i.e., stimuli) provides novel insights for understanding the relationship between neuronal activity and energetics. The courser spatial scale of fMRI (in sub µl range) relative to the electrical recordings (in sub nl range) necessitated comparison of CMRO2 /CMRO2 (derived from calibrated MRI) with the averaged ﬁring rate of a neuronal ensemble. Extracellular recordings respond to the activity of a small group of neurons in the vicinity of the electrode tip. Action potentials are easily identiﬁable signals in vivo because they are short lived, have relatively high SNR, and their different spike shapes can be assigned to individual neurons. Thus spike sorting from extracellular data (Zouridakis and Tam, 2000) have regional speciﬁcity to a few spiking neurons near the microelectrode tip located at layer 4 of the rat cortex in an activated region (guided by prior fMRI results). The spiking rate did not always increase with stimulation (Figure 10.8(C)). The nature of the ‘perturb and detect’ philosophy (e.g., see Kandel et al, 1991) usually biases experimentalists to focus on the population that showed an increase in ν upon stimulation onset. The criterion that allowed the inclusion of neuronal templates into the distributions (Figure 10.8(A) and (B)) was not whether the neuron responded to the stimulus or not, rather the condition was high SNR spikes. This approach revealed that a signiﬁcant fraction of the population (i.e., ∼30 % in condition II) preferentially did not respond to the stimulus. These results suggest that collaboration amongst a large number of neurons – some of which are ﬁring faster and others slower, but all requiring energy – is crucial for encoding information in the cerebral cortex (for a recent review see Scannell and Young, 1999). Since the spiking frequency distribution represents the different neurons in an ensemble, the changing of this distribution (Figure 10.8(A) and (B)) in conjunction with change in the total energy use (Figure 10.7(C)) symbolizes quantitative redeployment of the work by the ensemble. From a biophysical perspective, work is energy and vice versa (Garby and Larsen, 1995). Therefore the neuronal response of the ensemble at different workloads, which is depicted by the spiking frequency distributions in Figure 10.8(A)

Relationship between CMRO2 and Neuronal Activity (C)

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n (5 Hz)

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61 %

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0

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II

6

12 n (Hz)

18

24

n (10 Hz)

(B)

8%

t (50 s) n (5 Hz)

% Observations

t (100 s)

31 %

t (100 s)

Figure 10.8. Neural activity patterns of an ensemble. The neuronal activity patterns, as reﬂected by spiking frequency (ν), of a small ensemble (72 neurons) from the rat somatosensory cortex in α-chloralose-anesthetized rats for four different conditions: (A) two baseline conditions and (B) two stimulated conditions. Each bar in the histogram represents averaged value of ν for the baseline and stimulated conditions. The data for conditions I and II are shown in gray and black, respectively. There is a signiﬁcant difference between spiking frequency distributions in the two baseline conditions (p < 0.01), whereas upon stimulation the spiking frequency distributions become similar (p > 0.25). (C) Experimental variations across electrophysiological measurements (shown for condition II only) from the contralateral forelimb region. Signiﬁcant increases and decreases in ν were observed in 61 % and 8 % of the recordings, respectively (top and middle traces, respectively), whereas in 31 % of the recordings the stimulation did not induce any signiﬁcant changes in ν (bottom trace). The vertical and horizontal bars represent the scales for ν (in Hz) and time (in s), respectively. The thick black horizontal black bar represents the duration of the stimulus. All data adapted from Smith et al. (2002) and Hyder et al. (2002).

and (B), is commensurate with the total energies in each case (Figure 10.7(C)). The baseline comparison in Figure 10.8(A) shows that in condition II (deep anesthesia) the frequencies are lower than in condition I (light anesthesia), with a population at very low frequencies making a signiﬁcant contribution to the difference. In accordance with the similar total energies during stimulation from both baselines (Figure 10.7(C)), the histograms in Figure 10.8(B) show that not only do the average frequencies of the two states agree, but that the two distributions are very similar. This similar distribution has been reached by redeployment, not recruitment, of many neurons in the ensemble (Hyder et al., 2002). As shown in Figure 10.8(C) a large majority of neurons change their ﬁring rate upon stimulation – most increase the rate while a nonnegligible minority either decrease or do not appear to change their rates. The data clearly demonstrate that the changes in total energy (Figure 10.7(C)) are affected by widespread changes in neuronal ﬁring patterns of the ensemble (Figure 10.8(A) and (B)).

10.8. IMPLICATIONS FOR INTERPRETATION OF fMRI For technical reasons neuroimaging methods generally measure increments in local brain activity (for a review see Shulman et al, 2002). The ease of measuring incremental activity by functional imaging methods (with PET or fMRI) has been consonant with the widespread view in cognitive neuroscience that increments represent the modular energy required for function (Figure 10.9). Therefore the pertinent question is, does a cortical region require total or incremental neuronal activity for processing sensory information? If increments in CMRO2 and ν provide sufﬁcient support for the neural activity processing the sensory

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(C) Sensory input

(B)

Somatosensory cortex

Delocalized activity

Figure 10.9. A model of total neural activity to support cerebral function. Two possible scenarios of nonstimulated baseline (white) and stimulation-induced incremental (black) activities are shown in (A) and (B) respectively (Shulman et al., 1999; 2002). The jagged and vertical arrows represent the stimulation (same stimulus in all cases) and experimental parameters of neuronal activity are plotted vertically (same scale in all cases). (A) The stimulation induces the same increment in all cases independent of the baseline activity. In this case only the incremental activity contributes to information encoding. (B) The stimulation induces an increment which depends on the baseline, being larger from a lower baseline activity level. Here the total activity (i.e., baseline activity + incremental activity) is required for information encoding. (C) The microelectrode placement in layer 4 of the rat somatosensory cortex allowed measurement of the output from major excitatory efferents in the somatosensory cortex, i.e., the large pyramidal cells, which depend on connections from the thalamus (sensory input) as well as other regions of the cortex (delocalized activity). See Figure 10.3 for additional details about connections to and from layer 4. While the sensory input is the main choice of an experimental parameter to vary in neuroscientiﬁc studies, the delocalized activity can also be modiﬁed (e.g., by anesthesia dosage). The sum of the sensory and delocalized inputs, therefore, can modulate the total activity in a somatosensory region (Shulman et al., 2003). Future experiments in measuring either or both of these connections to the activated region in the cerebral cortex in greater detail (i.e., across different layers of the cortex) will provide greater insight into the interplay of population voting amongst the neuronal ensemble in sensing a particular stimulus.

stimulation, then these increments should be independent of the basal activity level (Figure 10.9(A)). This would allow the discard of the ‘baseline’ signal because the signal would not contribute to sensing of the stimulation. Alternatively, if the neuronal activity requires particular magnitudes of CMRO2 and ν, then their increments should be larger from deeper anesthesia (Figure 10.9(B)). In this case, however, the large and important ‘baseline’ signal cannot be discarded. The current results (Figure 10.7(C)) support the view that total activity is required for sensory processing. These results are in agreement with a survey from the literature comprising of CMRglc and spiking activity data (Shulman et al., 1999; 2002), which revealed that for sensory stimulus, the deeper the anesthesia the larger the increment, leading to the suggestion that the total energy rather than the increment was needed for function. To respond to the stimulus, the energy is raised from nonstimulated anesthetized levels to a total energy above or near the nonstimulated awake levels. The high levels of activity required for function have been supported by the present results, under the more reliable conditions of same animal, same stimulation, and the same anesthetic at different doses. Using energy as the measure of neuronal activity, the size of stimulus-induced incremental activity in awake human experiments is smaller than the basal activity (Shulman et al., 1999; 2002). In anesthetized animals the sizes of incremental and basal activities depend on depth of anesthesia and can be quite large (e.g., compare conditions I and II in Figure 10.7(C)). Therefore focus on only the magnitude of incremental activity neglects contributions of the basal activity, which can be misleading for interpretation of fMRI.

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Since the total activity in a neuroimaging voxel represents the integrated signal from proximal and distal neurons (Braitenberg and Schuz, 1991; Shepherd, 1994), the remote or delocalized neuronal connections from the rest of the brain can contribute to measures of total activity (Figure 10.9(C)). The total neuronal activity, which arises from both local and distal neurons, is reduced by anesthesia (Ueki et al., 1988; Hyder et al., 1996) both inside and outside the somatosensory region. However, upon stimulation the total neuronal activity inside the region of interest becomes high, and comparable to the activity at rest in the nonanesthetized awake condition (Shulman et al., 1999; 2002), whereas the total neuronal activity outside the stimulated region remains low (Ueki et al., 1992; Hyder et al., 1997). Thus forepaw stimulation introduces additional neuronal activity to the low baseline activity in the somatosensory cortex. However, in a nonanesthetized functional experiment the situation is slightly different. In the nonanesthetized awake brain at rest (Sokoloff et al., 1977), the forepaw stimulation may introduce a smaller additional neuronal activity thereby raising the total activity only slightly above the nonanesthetized awake resting state (Shulman et al., 1999; 2002). In this case, however, the fractional increase in the signal would be smaller. This type of interplay between contributions from proximal and distal neuronal connections may provide novel interpretation for fMRI as well as for brain organization (Changeux, 1985; Dehaene et al., 1998; Shulman et al., 2003). Although the measured neural activity in a somatosensory cortical site depends on the stimulation-induced sensory input (e.g., forepaw stimulation), the measured signal also depends on the delocalized signals from other regions of the brain (Figure 10.9(C)). Because the delocalized signals interact with the sensory input signals, the total measured signal in a region may potentially reveal insights into subjective experience (see Chapter 16). Because the baseline activity measured from layer 4 in the current studies (Figure 10.8(A)) also consists of signals from proximal and distal neurons (Braitenberg and Schuz, 1991; Shepherd, 1994), the large baseline activity (Figure 10.7(C)) could therefore be identiﬁed as input from and connections to other neurons including those in regions of the ‘global workspace’ (Dehaene et al., 1998). The widespread decrease of baseline activity in the anesthetized state must produce a decrease in global activity. Since anesthesia induces slow axonal conduction of most neurons (Richard, 1998), the reduction of global activity is consistent with the state of depressed activity during nonstimulated anesthetized states. Because the rest of the brain does not respond to forepaw stimulation and remains at low anesthetized activity (Ueki et al., 1988, 1992; Hyder et al., 1996, 1997), we propose that connections with the ‘global workspace’ are reduced during forepaw stimulation in the α-chloralose-anesthetized state. Furthermore under these anesthetized conditions, the localized total neuronal activity in the somatosensory cortex must include sizeable contributions from the vigorous sensory input. Thus total activities, as measured by experiments shown in Figures 10.5–10.8, provide quantitative measures that will help separate local from global responses.

10.9. BIOPHYSICAL BASIS OF COGNITIVE NEUROSCIENCE Cognitive neuroscientists use fMRI to create activation maps so that a ‘horizontal’ atlas can be created which can help locate regions of increased activity induced by speciﬁc sensory and psychological stimuli (Gazzaniga, 2000). This approach requires the subtraction of an important and required baseline activity, which as discussed above, removes an important part of the total activity that is required for cerebral function. What about the degree by which neuronal activity has changed? The current results (Figures 10.5–10.8) have allowed the quantiﬁcation of ‘vertical’ maps which can help provide information about the degree of neuronal activity changes induced by the stimuli. Because calibrated fMRI derives the fundamental parameter of neural energy (i.e., CMRO2 ) which in turn emulates neuronal work (i.e., ν), the total activity can be mapped noninvasively. In cases where the baseline is well deﬁned (e.g., sensory stimulation), differencing can localize responses with great validity and reliability based upon correlations with electrophysiological measures (e.g., SUA,

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MUA, or LFP). Cognitive modalities are difﬁcult to correlate, however, mainly due to the ambiguous deﬁnition of baseline in most tasks. There is little doubt, however, that differencing signals appropriately localize changes in some activities and the present results do not contradict these localizations (Posner and Raichle, 1998). However, meaningfulness of only using increments and discarding the important baseline signal is questioned (Shulman and Rothman, 1998) by the present results. The current ﬁndings challenge cognitive neuroscience studies, where it is generally assumed that incremental activity alone is responsible for signal processing. Therefore we propose that the goal of neuroimaging methods should be to measure both baseline and incremental signals in a quantitative manner (Hyder et al., 2002; Shulman et al., 2002). Although 13 C MRS methods can provide quantitative estimates of both signals (see Chapters 5 and 6) which represent activities of a very large and heterogeneous population of neurons, calibrated fMRI (see Chapter 9), which represents the grouped activity of the neuronal ensemble in the voxel (Figures 10.5–10.8), can be used to provide much higher spatial and temporal resolutions. Within the precision of the present measurements in which energy consumption and ﬁring rates increase proportionately, there are signiﬁcant contributions to the energy from quiescent neurons. Since about one third of the population (for condition II) did not respond to the stimulus (Figure 10.8(C)), the incremental energies during stimulation represent the distributed voting choices of the population of neurons (Figure 10.8(A) and (B)). The histograms of ﬁring rates depict the redeployment of the same neurons as a function of stimulated and nonstimulated conditions. While this redistribution of the population voting is measured as an increment by neuroimaging techniques (with PET or fMRI), the total energies of the neuronal population (Figures 10.7(C) and 10.9(B)) in the different conditions represent integrated activities of the populations, and the increments represent their shifts. The increment is only conceptually separable from the total activity by comparing total population activities in the different conditions. The neurons in an ensemble – with an averaged spiking frequency – when redeployed by different stimuli can be reﬂected by changes in the spiking frequencies (i.e., the neuroimaging signal can increase, decrease, or remain unchanged). However, at this time the variations in neuronal ﬁring patterns upon stimulation do not provide an understanding of the single neuron’s contributions to the entire ensemble, because we do not know exactly why the frequency of a speciﬁc neuron increases while for another it decreases. We can conclude, however, that the neurons that have been sampled within the somatosensory cortex are part of a local network of neurons which by anatomical necessity change their functional workload during forepaw stimulation (Harding et al., 1979). While distributions of ﬁring rates during nonstimulated conditions vary, the distributions of ﬁring rates during a particular stimulation in these limited experiments seem to be characteristic of that state, thus allowing the possibility that a particular population is a consequence of the same total energy being required during a particular stimulation, regardless of the starting baseline activity.

10.10. FUTURE DIRECTIONS While the collective activity of an ensemble of neural cells helps maintain normal cerebral function, glucose oxidation provides most of the energy needed for work (Siesjo, 1978). The term ‘work’ is deﬁned here as the sum of energy consuming processes associated with neuronal activity. The energetic demands of cellular work are extremely high (Chapter 7). Furthermore the localized changes, induced by depths of anesthesia and/or stimulations, of CMRO2 and ν in layer 4 of the somatosensory cortex were proportional (Figures 10.5–10.8). The ensemble activity in layer 4 depends on both the sensory input by the thalamus and the global input from other cortical regions. If sensory and delocalized inputs to total activity are deﬁned as task and baseline portions of the neuroimaging signal, then the current experimental results (Figures 10.5–10.8) have measured the total activity and have begun to separate sensory and delocalized contributions (Figure 10.9). Because delocalized signals (e.g., modiﬁed by anesthetic parameters) interact with the sensory inputs (e.g., modiﬁed by stimuli parameters), the total measured signal in a region may potentially reveal insights into the subjective experiences (Chapter 16).

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Since the activities that we have measured (Figures 10.5–10.8) arise from layer 4 neurons in response to speciﬁc anesthetics and stimuli, we need far more detailed characterization of sensory and delocalized inputs of total activity from other cortical layers and regions. In the future, detailed studies of neuronal populations in nonhuman primate models (Desimone, 1996; Goldman-Rakic, 1996; Tanaka, 1996; Georgopoulos, 2000; Logothetis, 2000; Rolls, 2000) will be needed for extensions to cognitive function in the awake human brain. Additional rat experiments are also needed in the future because the contributions of individual neurons in the voting population need to be evaluated more elaborately during stimulated and nonstimulated conditions for different experimental settings and/or paradigms. There are basically three experimental parameters that are ﬂexible in these types of anesthetized rat experiments; we can decrease the anesthesia level; we can change the stimulation; we can change the cortical depth of microelectrode penetration. By manipulation of these parameters in anesthetized rat experiments we would be able to extend the current results so that the total measured activity can be represented in terms of sensory and delocalized signaling. The ﬁrst goal would be to test whether the same total activity is reached during stimulation with different anesthetics (e.g., urethane, pentobarbital, propofol, halothane) at varying doses. In addition a necessary test would be whether altering the sensory stimuli (e.g., different frequencies and amplitudes) results in similar levels of activity from different baselines. While multiple microelectrode penetrations in an array-form (e.g., 12 × 12) to probe simultaneous activity of an ensemble (Szabo et al., 2001; Jog et al., 2002) is an option (Chicurel, 2001) for these future studies, this approach samples too few cells (e.g., less than a hundred) across very large regions (e.g., several millimeters), and most importantly it is much more invasive a process than penetration of a single microelectrode. Instead, sampling the electrical signals from multiple layers simultaneously with tetrodes (Gray et al., 1995; Hetherington and Swindale, 1999) that are vertically oriented may be a better option, although these electrodes would require careful development for these speciﬁc applications. There is, of course, the appeal for measuring neuroimaging signals with very high temporal resolution with the expectation that the fast signals will reveal new insights into brain function (Llinas, 2002). To this end, however, new technologies combining high ﬁeld fMRI with electrophysiology and/or optical imaging would play important roles. But these studies would be better suited for animal models. A much needed future research direction, however, is to test these proposals from the animal experiments on humans. Previous PET measurements in humans have reported that CMRglc decreases with depth of anesthesia (for a recent review see Heinke and Schwarzbauer, 2002). Although these reductions are global, small regional differences are also detected. Depression of glucose CMRglc (e.g., with isoﬂurane, halothane, and propofol) to the level of unresponsiveness (Alkire, 1998; Alkire et al., 2000; Heinke and Schwarzbauer, 2002) is related to reduced synaptic activity, as judged by various EEG variables as observed in anesthetized studies in animals (Shulman et al., 1999, 2002; Hyder et al., 2002). However functional activation studies under awake and anesthetized conditions in the human are greatly lacking.

10.11. CONCLUSION In neuroimaging (with PET or fMRI) incremental signals are generally measured. The subtraction of baseline activity removes an important part of the total activity (Shulman and Rothman, 1998) because most neurons in the ensemble contribute differently during the baseline and stimulation periods (Shulman et al., 2002). Thus quantitative interpretation by which neuronal activity has changed cannot be made from differencing. Prior 13 C MRS measurements in mammalian brain (Rothman et al., 1999) have revealed that the large energy utilization at basal conditions is almost exclusively used to support events associated with neurotransmission (Shulman and Rothman, 1998). The current results (Smith et al., 2002; Hyder et al., 2002) show the necessity of retaining that baseline signal in order to understand the activities of the neuronal ensemble for the different conditions. The total neuronal activity depicted by the distribution of neuronal spiking frequencies in a localized region, which is neither measured nor desired in neuroimaging,

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is conveyed by measuring total energy with calibrated fMRI (Hyder et al., 2001). We have integrated our results of both the incremental and baseline signals into a neuroscientiﬁc framework (Shulman et al., 2003), which describes participation of large neuronal populations during function. Since the total measured activity in a small region of the cortex represents the integrated signal from proximal and distal neurons, the delocalized signals from other regions (representing the ‘subjective’ contributions) can also modulate the localized signals from sensory stimuli (representing the ‘objective’ contributions). Thus interactions between sensory cues and the high basal activity will, in the near future, allow a more comprehensive view of the functioning brain. However, to achieve that with neuroimaging methods, the cognitive neuroscience community has to acknowledge the need for both the incremental and baseline signals.

REFERENCES Alkire MT (1998) Quantitative EEG correlations with brain glucose metabolic rate during anesthesia in volunteers Anesthesiology 89: 323–333 Alkire MT, Haier RG, Fallon JH (2000) Toward a uniﬁed theory of narcosis: Brain imaging evidence for a thalamocortical switch as the neurophysiologic basis of anesthesia – induced unconsciousness Conscious Cogn 9: 370–386 Ames III A (2000) CNS energy metabolism as related to function Brain Res Rev 34: 42–68 Bonvento G, Charbonne R, Correze JL, Borredon J, Seylaz J, Lacombe P (1994) Is alpha-chloralose plus halothane induction a suitable anesthetic regimen for cerebrovascular research? Brain Res 665: 213–221 Braitenberg V, Schuz A (1991) Anatomy of the Cortex: Statistics and Geometry. Springer, New York Brinker G, Bock C, Busch E, Krep H, Hossmann Ka, Hoehn-Berlage M (1999) Simultaneous recording of evoked potentials and T2 ∗ -weighted MR images during somatosensory stimulation of rat Magn Reson Med 41: 469–473 Castro-Alamancos MA, Connors BW (1997) Thalamocortical synapses Prog Neurobiol 51: 581–606 Changeux JP (1985) Neuronal Man: The Biology of Mind. Princeton University Press, Princeton, NJ (translated by Garcy L) Chicurel M (2001) Windows on the brain Nature 412: 266–268 Creutzfeldt OD (1975) Neurophysiological correlates of different functional states of the brain. In Alfred Benzon Symposium VII, Eds Ingvar DH, Lassen NA. Academic Press, New York, pp. 21–46 Dehaene S, Kerszberg M, Changeux JP (1998) A neuronal model of a global workspace in effortful cognitive tasks Proc Natl Acad Sci USA 95: 14529–14534 Desimone R (1996) Neural mechanisms for visual memory and their role in attention Proc Natl Acad Sci USA 93: 13494–13499 Dudley RE, Nelson SR, Samson F (1982) Inﬂuence of chloralose on brain regional glucose utilization Brain Res 233: 173–180 Garby L, Larsen PS (1995) Bioenergetics: Its Thermodynamic Foundations. Cambridge University Press, Cambridge, UK Gazzaniga MS (2000) The New Cognitive Neurosciences, 2nd edn. MIT Press, Cambridge, MA Georgopoulos AP (2000) Neural aspects of cognitive motor control Curr Opin Neurobiol 10: 238–241 Goldman-Rakic PS (1996) Regional and cellular fractionation of working memory Proc Natl Acad Sci USA 93: 13473–13480 Gottlieb JP, Keller A (1997) Intrinsic circuitry and physiological properties of pyramidal neurons in rat barrel cortex Exp Brain Res 115: 47–60 Gray CM (1995) Synchronous oscillations in neuronal systems: Mechanisms and functions J Comput Neurosci 1: 11–38 Gray CM, Maldonado PE, Wilson M, McNaughton B (1995) Tetrodes markedly improve the reliability and yield of multiple single-unit isolation from multi-unit recordings in cat striate cortex J Neurosci Methods 63: 43–54 Harding GW, Stogsdill RM, Towe AL (1979) Relative effects of pentobarbital and chloralose on the responsiveness of neurons in sensorimotor cerebral cortex of the domestic cat Neuroscience 4: 369–378 Heinke W, Schwarzbauer C (2002) In vivo imaging of anesthetic action in humans: Approaches with position emission tomography (PET) and functional magnetic resonance imaging (fMRI) Br J Anaesth 89: 112–122

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Ritchie JM (1967) The oxygen consumption of mammalian non-myelinated nerve ﬁbres at rest and during activity J Physiol 188: 309–329 Rolls ET (2000) Functions of the primate temporal lobe cortical visual areas in invariant visual object and face recognition Neuron 27: 205–218 Rothman DL, Sibson NR, Hyder F, Shen J, Behar KL, Shulman RG (1999) In vivo nuclear magnetic resonance spectroscopy studies of the relationship between the glutamate-glutamine neurotransmitter cycle and functional neuroenergetics Philos Trans R Soc London, Ser B 354: 1165–1177 Savaki HE, Desban M, Glowinski J, Besson MJ (1981) Local cerebral glucose consumption in the rat. I. Effects of halothane anesthesia J Comput Neurol 213: 36–45 Scannell JW, Young MP (1999) Neuronal population activity and functional imaging Proc R Soc London, Ser B Biol Sci 266: 875–881 Shepherd GM (1994) The Synaptic Organization of the Brain. Oxford University Press, New York Shulman RG (1996) Interview with Robert G Shulman J Cogn Neurosci 8: 474–480 Shulman RG (2001) Functional imaging studies: linking mind and basic neuroscience Am J Psychiatry 158: 11–20 Shulman RG, Rothman DL (1998) Interpreting functional imaging studies in terms of neurotransmitter cycling Proc Natl Acad Sci USA 95: 11993–11998 Shulman RG, Rothman DL, Hyder F (1999) Stimulated changes in localized cerebral energy consumption under anesthesia Proc Natl Acad Sci USA 96: 3245–3250 Shulman RG, Hyder F, Rothman DL (2002) Biophysical basis of brain activity: Implications for neuroimaging Q Rev Biophys 35: 287–325 Shulman RG, Hyder F, Rothman DL (2003) Cerebral metabolism and consciousness C R Biol 326: 253–273 Siesjo BK (1978) Brain Energy Metabolism. Wiley, New York Smith AJ, Blumenfeld H, Behar KL, Rothman DL, Shulman RG, Hyder F (2002) Cerebral energetics and spiking frequency: The neurophysiological basis of fMRI Proc Natl Acad Sci USA 99: 10765–10770 Sokoloff L (1991) Relationship between functional activity and energy metabolism in the nervous system: Whether, where and why?. In Brain Work and Mental Activity, Eds Lassen NA, Ingvar DH, Raichle ME, Friberg L. Munksgaard, Copenhagen, pp. 52–64 Szabo I, Czurko A, Csicsvari J, Hirase H, Leinekugel X, Buzsaki G (2001) The application of printed circuit board technology for fabrication of multi-channel micro-drives J Neurosci Methods 105: 105–110 Tanaka K (1996) Inferotemporal cortex and object vision Annu Rev Neurosci 19: 109–139 Thomson AM, Deuchars J (1997) Synaptic interactions in neocortical local circuits: Dual intracellular recordings in vitro Cerebral Cortex 7: 510–522 Ueki M, Linn F, Hossman KA (1988) Functional activation of cerebral blood ﬂow and metabolism and after global ischemia of rat brain J Cereb Blood Flow Metab 8: 486–494 Ueki M, Mies G, Hossmann KA (1992) Effect of α-chloralose, halothane, pentobarbital and nitrous oxide anesthesia on metabolic coupling in somatosensory cortex of rat Acta Anaesthesiol Scand 36: 318–322 Venkatasubramanian PN, Shen YJ, Wyrwicz AM (1996) Characterization of the cerebral distribution of general anesthetics in vivo by two-dimensional 19 F chemical shift imaging Magn Reson Med 35: 626–630 Winﬁeld DA, Gatter KC, Powell TP (1980) An electron microscopic study of the types and proportions of neurons in the cortex of the motor and visual areas of the cat and rat Brain 103: 245–258 Woolsey TA (1996) Barrels: 25 years later Somatosens Mot Res 13: 181–186 Yang X, Hyder F, Shulman RG (1997) Functional MRI BOLD signal coincides with electrical activity in the rat whisker barrels Magn Reson Med 38: 874–877 Zouridakis G, Tam DC (2000) Identiﬁcation of reliable spike templates in multi-unit extracellular recordings using fuzzy clustering Comput Methods Programs Biomed 61: 91–98

Section C Clinical Beginnings

11 NMR Studies of Bioenergetic Impairment in Human Epilepsy Hoby P. Hetherington Department of Radiology, The Albert Einstein College of Medicine, Bronx, NY, USA

Jullie W. Pan Department of Neurology, The Albert Einstein College of Medicine, Bronx, NY, USA

Dennis D. Spencer Department of Neurosurgery, Yale University, New Haven, CT, USA

11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8

Introduction H Spectroscopic Localization and Lateralization of Epileptogenic Regions Factors Affecting Sensitivity and Speciﬁcity Interpretation of Decreases in NAA Levels 31 P Spectroscopic Measurements of Bioenergetics in Human Epilepsy Factors Affecting the Interpretation of 31 P Data Interpretation of the Bioenergetic Alterations Possible Mechanism Linking Bioenergetic Impairment and Seizures 1

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11.1. INTRODUCTION Epilepsy is one of the most common neurological disorders, affecting one to two million Americans (1). For approximately 10 % of these individuals, the debilitating effects of the disease cannot be controlled Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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by medication. In this population surgical intervention can be highly effective, provided there is accurate lateralization and localization of the epileptogenic tissue. Over the past decade, a variety of methods for lateralizing and localizing seizure foci, MR imaging (2–5), PET (6–13), and MR spectroscopy (13,14–19) have been used. Although these measures have often been used simply as surrogate markers for the presence of disease, the alterations, when viewed in the context of the biochemical information present, provide a unique insight into the biochemical pathophysiology of the disease. Unlike conventional MRI studies, which detect the concentration and cellular environment of tissue water, MR spectroscopy provides speciﬁc biochemical and histochemical information. For example, 1 H spectroscopic measurements have utilized decreases in N -acetyl aspartate (NAA), a compound synthesized only in neuronal mitochondria, to identify regions of neuronal injury (13–19). Increases in total creatine (both phosphorylated and nonphosphorylated forms) and choline have been interpreted as reﬂecting gliosis and altered membrane turnover. In temporal lobe epilepsy, where neuronal loss and reactive gliosis is a common ﬁnding, decreases in NAA and or increases in creatine and choline have been shown to have lateralization sensitivities as has high as 97 % (20). Using 31 P spectroscopy, in vivo concentrations of phosphocreatine, ATP and inorganic phosphate can be imaged noninvasively along with intracellular pH to provide a measure of bioenergetic status. Interestingly, despite the reduction in metabolic rate during the interictal period commonly seen in FDG PET studies, alterations in bioenergetics (decreases in phosphocreatine/ATP (PCr/ATP) and PCr/inorganic phosphate (PCr/Pi)) have emerged as a consistent ﬁnding for a variety of epilepsies (21–26). Taken together these ﬁndings suggest a common pathophysiology of impaired neuronal bioenergetics underlying a variety of different epilepsies. In this chapter we will review the role that MR spectroscopy has had as a clinical tool in identifying epileptic foci. As part of this discussion, we will also identify and discuss some of the technical hurdles that must be accounted for in acquiring and accurately interpreting in vivo MR spectroscopy data. Speciﬁcally we will discuss the effects of metabolite heterogeneity as a function of tissue type and how a failure to account for this can result in signiﬁcant artifacts that degrade the interpretability of the data. As part of this discussion we will also discuss several approaches to overcome these effects. We will then discuss the biological signiﬁcance of these ﬁndings and how they suggest that bioenergetic impairment occupies an important role in the pathophysiology of seizure disorders. Finally we will discuss a possible model that links the metabolic changes and energetic impairment seen by spectroscopic studies to the primary clinical manifestation of epilepsy, inappropriate spontaneous electrical activity.

11.2. 1 H SPECTROSCOPIC LOCALIZATION AND LATERALIZATION OF EPILEPTOGENIC REGIONS During the mid 1990s a number of groups demonstrated the utility of 1 H spectroscopic measurements of NAA in the lateralization of the epileptogenic region in patients with temporal lobe epilepsy (14–19). Speciﬁcally, changes in NAA content (14), NAA/Cr (17–19), NAA/CH (16) and NAA/(Cr + CH) (15) have all been used to lateralize the seizure focus in patients with intractable temporal lobe seizures. Based on the localization of NAA to neurons (8,27) and the ﬁnding that in cell cultures that Cr is higher in glial cells (28), these changes were initially interpreted as representing a combination of neuronal loss (NAA decreases) and glial proliferation (Cr increases). This interpretation was consistent with the known pathophysiology of temporal lobe epilepsy where both neuronal loss and gliosis are common. As a lateralization and localization tool, the reported sensitivities have ranged from 60 % (15) to 97 % (20), somewhat higher than the 74 % typically reported for conventional MRI studies (29). For example Chu et al. (30) reported that MR spectroscopic imaging when combined with tissue segmentation correctly lateralized the epileptogenic hippocampus in patients without MRI detectable volumetric loss. This is despite the 300-fold decrease in volume resolution typical for spectroscopy studies in comparison

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to conventional MRI studies. The ability of MR spectroscopy to attain higher sensitivities in comparison to standard imaging is most likely the result of the following factors: (1) that the metabolic perturbation seen in temporal lobe epilepsy is widespread, affecting not only the hippocampus (3–4 cm3 in volume) but also surrounding temporal gray and white matter, and (2) that detectable decreases in hippocampal volume by conventional MRI may require neuronal losses of up to 50 %. Thus for temporal lobe epilepsy, where the effects of the disease are widespread and submillimeter spatial resolution is not required, the biochemical speciﬁcity afforded by spectroscopy provides signiﬁcant advantages over higher spatial resolution measurements of tissue water. In addition to providing a highly sensitive method for lateralizing the seizure focus, spectroscopic imaging studies may also provide some predictive insight as to surgical outcome. In these spectroscopy studies (18,20,30,31), 40–50 % of the subjects studied and diagnosed as having unilateral temporal lobe epilepsy, based on EEG studies, were found to have signiﬁcant decreases in NAA in the contralateral hemisphere. These data indicated that, although the seizures were found to predominantly emanate from one hippocampus, the effects were clearly manifesting also in the contralateral hemisphere. Kuzniecky et al. (32) correlated the severity of NAA/Cr alterations in the ipsilateral and contralateral hippocampi with surgical outcome. Although the severity of NAA/Cr abnormality in the ipsilateral hippocampus did not appear to have a signiﬁcant impact on surgical outcome, more severe alterations in the contralateral hippocampus were associated with poorer surgical outcomes, i.e. continuing seizures. Whether this ﬁnding reﬂected the presence of small (below the detection of MRI) but signiﬁcant neuronal loss in the contralateral hippocampus or an ongoing biochemical defect could not be resolved. The ﬁnding of decreased NAA is not solely restricted to temporal lobe epilepsy. In particular, reduced NAA levels have been reported in a variety of epileptic subtypes including frontal lobe epilepsy (33), malformations of cortical development (34,35), and Rasmussen’s encephalitis (36). Thus the use of 1 H spectroscopic imaging for the lateralization and localization of the epileptic foci is of general use in the lateralization and localization of epileptogenic foci.

11.3. FACTORS AFFECTING SENSITIVITY AND SPECIFICITY The sensitivity and speciﬁcity of these studies ranged from 60 % (15) to 97 % (20), with greater sensitivity and speciﬁcity reported from groups using higher spatial resolution spectroscopic imaging techniques. These reports suggested that metabolic heterogeneity and/or partial volume effects could play a signiﬁcant role in determining the accuracy and efﬁcacy of the measurements. Quantitative large single voxel measurements from occipital gray matter and parietal white matter (37,38) demonstrated tissue differences between white and gray matter contents of creatine (Cr) (6.3, 8.3 mM), N -acetyl aspartate (NAA) (10.3, 10.7 mM) and choline (1.8, 1.5 mM) (Figure 11.1). This was subsequently conﬁrmed in spectroscopic imaging studies using 0.5 ml volumes at 4 T and image segmentation (39), reporting values for Cr (6.1, 8.2 mM), NAA (10.5, 9.7 mM) and choline (1.5, 1.5 mM). Based upon these observations it became clear that tissue heterogeneity could be a limiting factor in the identiﬁcation of metabolically altered regions. Speciﬁcally, due to the lower creatine content in white matter as opposed to gray matter, inclusions of signiﬁcant amounts of white matter in predominantly gray matter voxels would lower total creatine content and increase the NAA/Cr ratio, thereby mimicking more healthy tissue resulting in false negatives. Similarly, volumes with substantial gray matter, due to the higher creatine content would artiﬁcially bias the NAA/Cr ratio downwards, giving the appearance of greater damage. Additionally, it has been demonstrated that the NAA/Cr ratio in the cerebellar vermis (found medial and inferior to the hippocampus) is very low (0.72) in comparison to temporal gray and white matter (1.51, 1.79) (18). Combined, both of these effects can result in false positives and false negatives complicate the lateralization of the epileptogenic region. To overcome the effects of tissue heterogeneity, a number of investigators in a variety of different disease states, have employed linear regression analyses to compensate for the inclusion of varying levels of gray

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and white matter. Although these methods are most commonly used in global pathologies to identify gray or white matter speciﬁc alterations in comparison to control subjects (40,41), they can also be employed on a voxel by voxel basis within subjects (30,39). Using segmented anatomical images, the gray and white matter content of each spectroscopic imaging voxel can be determined. From these data a regression analysis can be performed in healthy volunteers to obtain the ‘pure’ gray and white matter metabolite contents and ratios (Figure 11.2(A)). Using the population statistics of this data, the probability that any voxel of speciﬁc gray and white matter content in a patient study is abnormal can be calculated using

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Equation (11.1) (39). Cr/NAA(p ≤ α) = Cr/NAAmean + tα,n−2 {MSE [1 + 1/n + (xgm,mean − xgm )2 /Sxx ]}1/2

(11.1)

where Cr/NAA(p ≤ α): the cutoff value corresponding to a p value ≤ α for Cr/NAA, Cr/NAAmean : the expected value of the Cr/NAA ratio for xgm,mean content of gray matter, tα,n−2 : t value for p ≤ α for n − 2 degrees of freedom, n: the number of pixels, MSE : the residual mean square, xgm is the fraction of gray matter of the voxel, xgm,mean is the mean fraction of gray matter from the population. Voxels that are statistically abnormal (p < 0.05) can then be color coded according to p value and superimposed on the anatomical images for easy evaluation and interpretation (Figure 11.2(B)). Chu et al. (30) have demonstrated that when these regression-based statistical methods with image segmentation are utilized in patients with temporal lobe epilepsy, false negatives and incorrect lateralizations are reduced in comparison to simple left–right voxel comparisons. Of note in this particular patient group, fully one-half of the patients could not be lateralized by conventional MRI methods, due to either an absence of hippocampal atrophy or the presence of bilateral atrophy. Thus in these cases, MR spectroscopy provided signiﬁcant new information not available in the clinical MRIs. As described, heterogeneity due to gray and white matter mixing can be accounted for by using image segmentation schemes and linear regression-based statistical analyses. However, this assumes that there is no regional variation within the gray or white matter itself. Recently Vermathen et al. (42) have reported that NAA levels show an anterior–posterior bias along the hippocampal formation, with higher levels reported from more posterior locations, while choline and creatine showed the opposite trend. Thus additional corrections for local heterogeneity may also be required.

11.4. INTERPRETATION OF DECREASES IN NAA LEVELS Based on the localization of NAA to neurons (27,28), the reduced NAA content found in patients with temporal lobe epilepsy was initially interpreted as being the result of neuronal loss. However, the report of Hugg et al. (43) of NAA recovery from the contralateral hippocampus in patients with bilateral abnormalities 1 year after successful surgery suggested that at least a portion of the observed NAA loss was not solely due to neuronal loss. This ﬁnding was conﬁrmed by Cendes et al. (44) and Vermathen et al. (45). In a follow-up study, on average, 50 % of the metabolic recovery occurred over a period of less than 6 months, demonstrating a slow but responsive nature of neuronal NAA levels (46). This indicated that alterations in NAA were not solely due to neuronal loss. In epilepsy patients with malformations of cortical development, signiﬁcant reductions were also observed in NAA/Cr in the epileptogenic region (34,35). In these patients, NAA alterations were seen in the absence of neuronal loss (34). Speciﬁcally the resected tissue showed normal or elevated numbers of neurons, although morphologically they were abnormal (34). Subsequently Kuzniecky et al. (47) demonstrated that NAA losses in patients with temporal lobe epilepsy were also not signiﬁcantly correlated with neuronal and glial densities. Thus in both temporal lobe epilepsy and malformations of cortical development, the reductions in NAA reﬂect a process which is not solely due to neuronal loss, but somehow reﬂects the functional status of the neurons themselves. To probe the histological basis for the alterations in NAA, we correlated in vivo measurements of NAA from the hippocampi of patients with intractable TLE with quantitative measurement of neuronal loss and reactive astrocyte proliferation in the resected tissue. Proliferation of reactive astrocytes, as expressed by

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the presence of glial ﬁbrillary acidic protein (GFAP) has been associated with recent or ongoing neuronal injury (48). Regression analyses of NAA/Cr measured in vivo with quantitative neuronal counting across CA1–CA4 and the dentate gyrus revealed statistically signiﬁcant correlations only in the CA2 sector (Figure 11.3(A)). Interestingly, a similar correlation between GFAP staining with neuronal number revealed statistically signiﬁcant correlation again only in the CA2 sector of the hippocampus (Figure 11.3(B)). However, a statistically signiﬁcant correlation was found between NAA/Cr and GFAP staining across all four sectors (Figure 11.3(C)). This suggests that in temporal lobe epilepsy, reductions in NAA/Cr more closely correlate with the presence of reactive astrocytes than alterations in neuronal density. The presence of reactive astrocytes is largely interpreted as a marker for recent or ongoing neuronal injury. Interestingly, neuronal loss in the CA1, CA3, and dentate gyrus is typically believed to be largely due to the initial

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precipitating insult in temporal lobe epilepsy, while neuronal loss in the CA2 is more generally believed to correlate with the progression of the disease (49). The reversibility of decreased NAA in the contralateral hippocampus with successful surgical outcome, i.e. cessation of seizures, suggests that the recovering NAA levels reﬂect a ‘healing’ or ‘normalization’ of cellular process in the contralateral hippocampus. The fact that these processes are associated with ‘normalization’ of neuronal function, i.e. cessation of seizures, suggests that the processes, which link the balance between NAA synthesis and degradation, may also be linked with neuronal function. Despite the extensive use of NAA as a marker for neuronal injury in a wide variety of disorders including stroke (50), multiple sclerosis (51), Alzheimer’s disease (52) and AIDS dementia (53), its biological function remains controversial. NAA has been reported to act as a source of acetate transfer from the mitochondria to the cytoplasm for lipid synthesis (54). Additionally, NAA has been proposed to act as a transporter of amino nitrogen from the mitochondria to the cytoplasm (55). NAA has also been proposed to act as an osmotic agent, based on its redistribution between extracellular/intracellular compartments in response to osmotic stress (56). Investigations of agents that perturb mitochondrial function have been demonstrated to affect NAA levels and production rates. Heales et al. (57) used l-buthionine sulfoximine administration to deplete glutathionine resulting in cytochrome c oxidase activity decreases of 27 %, and citrate synthase activity of 18 %. Accompanying these changes, NAA levels were decreased 11 %. Bates et al. (58) used rotenone, cyanide and oligomycin in isolated rat brain mitochondria. In these cases ATP production, oxygen consumption and NAA production were positively correlated and decreased relative to control preparations. Thus the decreased NAA levels seen in epileptics may in fact represent alterations in the function of neuronal mitochondria and their role in modulating seizure activity. This is also consistent with the ﬁnding that in epilepsy patients with cortical malformations of development who are well controlled (less than four seizures year), NAA levels are not altered despite the presence of structural abnormalities (34). If true, this would suggest that bioenergetic deﬁcits should be present in the hippocampi of patients with temporal lobe epilepsy and that upon successful treatment these values should normalize.

11.5.

31

P SPECTROSCOPIC MEASUREMENTS OF BIOENERGETICS IN HUMAN EPILEPSY

Prior to the widespread availability of 1 H spectroscopy on clinical MR systems, Weiner and colleagues evaluated the use of 31 P spectroscopy for the lateralization of epileptogenic regions. In these initial studies at 2.0 T, signiﬁcant alterations in inorganic phosphate (73 % increase) and brain pH (0.17 unit increase) were reported (21,22). Although subsequent studies at 1.5 T and 4.1 T failed to reproduce the reported changes in pH, signiﬁcant alterations in bioenergetic parameters were reported (23,24). At 1.5 T Kuzniecky et al. reported that PCr/Pi was reduced by 50 % in the ipsilateral temporal lobe whereas it was reduced by 24 % in the contralateral lobe (23). At 4.1 T, Chu et al. (24) later reported that PCr/Pi was signiﬁcantly decreased in both the ipsilateral (32 %) and contralateral lobes (19 %). Within this group, 73 % of the patients were correctly lateralized using the PCr/Pi ratio. Despite the controversy regarding pH changes, bioenergetic impairment (increased Pi and decreased PCr/Pi) was reported by both groups. Since patients with recent seizures (less than 24 h previous) were excluded, these effects are believed not to be an acute effect of seizures, but reﬂect a chronic impairment of energy metabolism, consistent with a mitochondria-based deﬁcit leading to NAA reductions. Declines in bioenergetics are also seen in a variety of other forms of epilepsy including children with Lenox–Gasteau syndrome (25), absence seizures (25) and in adults with frontal lobe epilepsy (26). In children with Lenox–Gasteau and absence seizure, a nonfocal form of epilepsy, PCr/ATP was decreased in the cortical gray matter in comparison to control subjects. Notably, no such change was seen in white

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matter, reﬂecting the speciﬁcity to neuronal bodies and their high energetic demands. In patients with frontal lobe epilepsy, Garcia et al. (26) reported elevations of inorganic phosphate, consistent with a decrease in overall energy charge. Thus the ﬁnding of bioenergetic impairment is not restricted solely to temporal lobe epilepsy, but may reﬂect a basic mechanism underlying epilepsy as a whole.

11.6. FACTORS AFFECTING THE INTERPRETATION OF

31

P DATA

Although 31 P studies have shown that abnormalities in bioenergetics are present their use to date has been limited. Part of the limited use of 31 P spectroscopy can be attributed to the large range of normal values for cerebral PCr (3–5 mM) and ATP (2–4 mM) in low ﬁeld studies (59–62), and the resulting perception that 31 P spectroscopy is highly inaccurate. These initial reports used large volumes of 25–100 cm3 and did not attempt to account for tissue or regional heterogeneity. Although spatial resolution for 31 P studies is limited by the SNR, recent studies even at high ﬁeld have identiﬁed clear regional heterogeneities in 31 P metabolites using regression analyses (63) (Figure 11.4). Although PCr levels are relatively uniform between cerebral gray and white matter and mixed cerebellar volumes (3.53 ± 0.33, 3.33 ± 0.37 and 3.75 ± 0.66 mM respectively), ATP levels show signiﬁcant heterogeneity ranging from 1.78 ± 0.56 mM in the mixed cerebellar volumes to 2.19 ± 0.33 and 3.41 ± 0.33 mM in cerebral gray and white matter Although skeletal muscle contributions to central volumes within the brain are negligible, due to the high concentration of PCr and ATP in temporalis muscles, (25.4 ± 2.3 and 8.5 ± 1.9 mM respectively), their contribution to the peripheral temporal regions can be substantial. The effect of this can be seen in the spectroscopic image of PCr/ATP shown in Figure 11.5. In this image we have overlaid a PCr/ATP image (B)

(A) cgm = 28% cwm = 67% csf = 5% cbl = 0% sm = 0%

(C)

cgm = 59% cwm = 34% csf = 8% cbl = 0% sm = 0%

(D) cgm = 17% cwm = 6% csf = 13% cbl = 64% sm = 0%

cgm = 13% cwm = 2% csf = 6% cbl = 0% sm = 23%

Figure 11.4. Spectra from predominantly (A) cortical white matter (CWM), (B) cortical gray matter (CGM), (C) the cerebellum (CBL) and (D) skeletal muscle (SM). The contribution of each tissue type, including cerebral spinal ﬂuid (CSF) is listed above each spectrum.

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R - ipsi

Figure 11.5. (Plate 4). A scout anatomical image and a composite image of the anatomical image and a color-coded overlay of PCr/ATP. The highest levels of PCr/ATP are colored red, while intermediate values are in yellow and light blue, while the lowest levels are in dark blue. The seizure focus (ipsi) was found to be on the right (left in the image) and is dark blue, while the contralateral hippocampus is light blue.

onto an anatomical image for reference. Higher PCr/ATP ratios are in red and yellow, with lower levels in blue. As can be seen the highest ratios are seen from the temporalis muscle, while the lowest values are seen in the white matter. In this case, the ipsilateral hippocampus (seizure origin) has a PCr/ATP value that is signiﬁcantly lower than the contralateral hippocampus and other gray matter regions such as occipital gray matter but is similar to ‘pure’ white matter. Thus failure to account for these differences can result in substantial variability that is not reﬂective of underlying pathology, but rather normal heterogeneity based on tissue composition. Using a ﬁve-compartment model including cerebral gray and white matter, skeletal muscle, cerebellar tissues and cerebral spinal ﬂuid, we have demonstrated this heterogeneity can be compensated for in the regions about the hippocampus and temporal lobe. Displayed in Figure 11.6 is a plot of the predicted and measured values for PCr and ATP from 22 voxels from the temporal region. Despite the two-fold variations in measured ATP and PCr content, the predicted values based on tissue composition are in good agreement throughout the range. Using this methodology, the decrement in PCr/ATP from the epileptogenic region can be calculated, correcting for varying degrees of tissue composition. Displayed in Figure 11.7, is a scout image and spectra selected from the inferior aspect of the hippocampus. As can be seen, the largest decrements in PCr/ATP are found overlying the ipsilateral hippocampus. Thus corrections for tissue heterogeneity are essential improve our ability to interpret 31 P spectroscopic imaging data of patients with temporal lobe epilepsy. Even after correction for tissue content differences, the large size of the spectroscopic imaging voxels for 31 P measurements, 6–12 cm3 , can result in substantial partial volume effects with surrounding healthy tissue. This effect can be compounded when the region of interest lies at an intersection of the spectroscopic imaging grid, thereby distributing the signal arising from the affected region into as many as eight voxels for a 3D study. Although spatial interpolation of the data set by zero ﬁlling can help to offset this problem, the resulting data sets become large (>1 GB for 1.5 mm spatial accuracy) resulting in signiﬁcant data handling problems. To overcome this effect, the reconstruction grid can be arbitrarily shifted post-acquisition using the 3D anatomical images to calculate the necessary phase shifts to allow precise centering of the

NMR Studies of Bioenergetic Impairment in Human Epilepsy PCr

207 ATP

1.5 × 106

Measured

Measured

1.5 × 106

0.0 × 106 0.0 ×

1.5 ×

106

0.0 × 106 0.0 × 106

106

Predicted

Predicted

1

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3

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7

5

1.5 × 106

6

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Figure 11.6. Plots of the predicted and measured values of PCr and ATP from 22 voxels (open squares) and (ﬁlled squares) from the region about the hippocampus. The solid line indicates values for which the measured and predicted values are identical. The locations of the 22 voxels (10 voxels spanning a row across the head at the level of the hippocampi and 12 spectra from a rectangular region about the hippocampi and superior portion of the cerebellum) are indicated on the scout images.

voxels of interest with 1.5 mm accuracy (64). Using the this method, the variation in gray and white matter content of the selected voxels was dramatically reduced, also reducing the measured variation in PCr/ATP, providing for greater accuracy in comparing speciﬁc anatomical locations between subjects and controls. The fractional content of gray matter in the selected volumes was 60 ± 5 %, 55 ± 4 % and 47 ± 4 % for the amygdala, pes and hippocampal body in controls. We then evaluated the extent of energetic impairment along the hippocampal formation (65). As shown in Figure 11.8, we observed a gradient of energetic impairment, with the greatest decline from anterior locations, and also greater declines in the ipsilateral hippocampus compared to the contralateral hippocampus. All three ipsilateral locations (body, pes and amygdala) were statistically different from normal controls, while the contralateral amygdala and pes were also statistically abnormal. Speciﬁcally, these data demonstrate that there is a clear regional heterogeneity in the impairment such that the decrement in PCr/ATP in comparison to normal controls is amygdala > pes > body and ipsi > contra. Analysis of the fractional tissue content of gray and white matter between patients and control subjects revealed no signiﬁcant differences for the same locations

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∆PCr/ATP= −0.28

6

2

∆PCr/ATP= −0.23

5

3

4

Figure 11.7. A scout image and spectra spanning the hippocampi in a patient with temporal lobe epilepsy. The calculated decrement in PCr/ATP is displayed above the spectra from the ipsilateral hippocampi.

A Contra

Ipsi

P

B

Figure 11.8. Orthogonal views from a 3D image set spanning the brain of a patient with temporal lobe epilepsy. The locations of the selected voxels are identiﬁed by the box and cross-hairs. The selected locations were: (A) amygdala; (P) pes, and (B) hippocampal body. The corresponding spectra from the ipsilateral and contralateral hippocampi are displayed in the spectra to the right.

allowing the locations to be directly correlated. The ﬁnding of greater energetic impairment from more anterior locations is also consistent with 1 H MRSI data of Vermathen et al. (45) who reported that NAA declines were also more pronounced anteriorly in temporal lobe epilepsy hippocampus. This again supports the hypothesis that the reductions in NAA reﬂect ongoing impairment that may be mitochondrial in origin.

11.7. INTERPRETATION OF THE BIOENERGETIC ALTERATIONS Similar to that seen in the 1 H studies, Simor et al. (66) using 31 P spectroscopic imaging reported that the energetic deﬁcits seen in temporal lobe epilepsy patients were also reversible with successful surgery. In

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this study, signiﬁcant improvements in the contralateral hippocampus were observed 1 year after successful surgery. This indicated that the alterations in 31 P metabolites were also not solely due to neuronal death, but reﬂected a dynamic and reversible process. Most recently, Pan et al. (25) have demonstrated that in pediatric epilepsy patients being treated with the ketogenic diet, bioenergetic impairments present prior to initiation of the diet are reversed as the seizures are brought under control. Speciﬁcally, after 4 months of the ketogenic diet therapy, the PCr/Pi and PCr/ATP ratios had improved by 22 and 14 % respectively. Thus both reductions in NAA and impairments in bioenergetics appear to be tightly linked with seizure activity and can be reversed with successful treatment. Thus it appears clear that there is a signiﬁcant bioenergetic component to the etiology of seizure disorders. However, it remains unclear as to whether this reﬂects the general damage associated with seizures or is a causative factor. If bioenergetic impairment is a causative factor, alternative treatments that modulate brain bioenergetics could provide an effective avenue for treatment. One such therapy is the use of the ketogenic diet in the treatment of nonlesional pediatric epilepsies. In this regimen, patients who are placed on a high fat, low carbohydrate diet become systemically ketotic, with elevated plasma and brain ketone levels. Although the exact mechanism for the ketogenic diet remains controversial, it is known that ketones can supplement or replace glucose as a fuel source for oxidation. In adults, ketone utilization has been reported to provide up to 25 % of total brain needs after only 3 days of fasting (67). Given that ketones are preferentially utilized by neurons as an alternate fuel source for oxidation (68), ketones can circumvent the normal mechanisms that couple glucose uptake to neuronal functional activity. Although the ketogenic diet is most commonly used in pediatric patients, recently Sirven et al. (69) have reported signiﬁcant success in adult patients who maintain ketosis while on the diet. This ﬁnding further strengthens the general role of metabolism and bioenergetics in the pathophysiology of human epilepsy. However, this does not provide a mechanism linking the bioenergetic impairment directly to the symptoms of the disease, inappropriate electrical activity.

11.8. POSSIBLE MECHANISM LINKING BIOENERGETIC IMPAIRMENT AND SEIZURES How these alterations in energetics and mitochondrial function may directly affect neurotransmission and the development of epilepsy is unknown. One possible mechanism is by modulating extracellular glutamate through alterations in Ca2+ sequestration in the pre-synaptic terminal. For example, Scotti et al. (70) have shown, in hippocampal cell cultures, that dissipation of the mitochondrial membrane potential with FCCP results in depolarization of the mitochondria and an inability of mitochondria in the pre-synaptic terminals to sequester intracellular Ca2+ . Elevations in Ca2+ in turn signal for release of synaptic vesicles and thus release the release of glutamate in glutamatergic terminals. As predicted, Scotti et al. (70) reported that the failure to sequester Ca2+ in turn led to increased spontaneous excitatory post-synaptic currents. Using the calyx of Held as a model for glutamatergic synapses, Billups et al. (71) reported that dissipation of the mitochondrial membrane potential with rotenone and cyanide p-triﬂuromethoxyphenylhydrazone results in elevations of intracellular Ca2+ , and concluded that mitochondria are the major organelle regulating pre-synaptic calcium at central glutamatergic terminals. Although cytoplasmic Ca2+ can also cleared by the Ca2+ ATPase and the Na+ /Ca2+ exchanger, impairments in oxidative metabolism (by decreasing the availability of ATP) can also reduce ﬂux through the Ca2+ ATPase. This would prolong elevations in Ca2+ , and thus enhance the release of glutamate. Similarly, a failure in oxidative metabolism might also reduce the efﬁciency of Na+ pumping and thus reduce the Na+ gradient across the terminal thereby depleting the driving force for the Na+ /Ca2+ exchanger. Thus in model systems (hippocampal tissue culture) neuronal mitochondrial damage and failures in neuronal bioenergetics can result in alterations in increased vesicular release of glutamate. As the primary excitatory

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amino acid in mammalian brain, increased extracellular glutamate concentrations have been linked with hyper-excitability in several animal seizure models. Thus impairments in oxidative metabolism in the neuronal mitochondria can lead to the build-up of extracellular glutamate, resulting in hyperexcitability and seizures. If true, the mitochondrial impairment would display the characteristic spectroscopic ﬁndings of decreased NAA and reduced PCr/ATP. Reversal of the mitochondrial impairment could restore extracellular glutamate homeostasis, which in principle could eliminate the inappropriate electrical activity. This too would manifest itself as an improvement in bioenergetics, as seem in the ketogenic diet work of Pan et al. (25), and the restoration of NAA levels as seen in temporal lobe epilepsy patients. Although the proposed mechanism is consistent with the spectroscopy data, additional investigations correlating intracellular Ca2+ and extracellular glutamate with 1 H and 31 P spectroscopic measures are needed to further evaluate the validity of these hypotheses. As described, we have focused on the possible role of impairments in neuronal oxidative metabolism as leading to elevations of extracellular glutamate through enhanced glutamate release, possibly mediated by Ca2+ . However, extracellular glutamate levels are determined through the balance of both release and reuptake mechanisms. Thus metabolic alterations that lead to reduced astrocytic reuptake of extracellular glutamate and glutamate–glutamine cycling can also lead to increased extracellular glutamate and hyperexcitability. This possible mechanism will be the topic of the next chapter.

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64. Twieg DB; Meyerhoff DJ; Hubesch B; Roth K; Sappey-Marinier D; Boska MD; Gober JR; Schaefer S; Weiner MW, Phosphorus-31magnetic resonance spectroscopy in humans by spectroscopic imaging: localized spectroscopy and metabolite imaging, Magn Reson Med 12: 291–305, 1989. 65. Hetherington HP; Pan JW; Firlik KS; Spencer DD, Bioenergetic abnormalities in temporal lobe epilepsy, Proc Int Soc Magn Reson Med p 419, 2002. 66. Simor T; Chu WJ; Hetherington HP; Kuzniecky RI; Elgavish GA, Tailored temporal lobectomy induced improvements in 4.1 T 31PNMR SI generated phosphorous metabolite indices in temporal lobe epilepsy, Proceedings of the International Society of Magnetic Resonance in Medicine, p 33, Vancouver, British Columbia, Canada, April 1997. 67. Hasselbalch SG; Knudsen GM; Jakobsen J; Hageman LP; Holm S; Paulson OB, Brain metabolism during shortterm starvation in humans, J Cereb Blood Flow Metab 14: 125–131, 1994. 68. Pan JW; de Graaf RA; Petersen KF; Shulman GI; Hetherington HP; Rothman DL, [2,4-13C2]-b-hydroxybutyrate metabolism in human brain, J Cereb Blood Flow Metab 22: 890–898, 2002. 69. Sirven J; Whedon B; Caplan D; et al., The ketogenic diet for intractable epilepsy in adults: preliminary results, Epilepsia 40: 1721–1726, 1999. 70. Scotti AL; Chatton JY; Reuter H, Roles of Na(+)–Ca2+ exchange and of mitochondria in the regulation of presynaptic Ca2+ and spontaneous glutamate release, Philos Trans R Soc London Ser B Biol Sci 354(1381): 357–364, 1999. 71. Billups B; Forsythe ID, Presynaptic mitochondrial calcium sequestration inﬂuences transmission at mammalian central synapses, J Neurosci 22(14): 5840–5847, 2002.

12 MRS Studies of the Role of Altered Glutamate and GABA Neurotransmitter Metabolism in the Pathophysiology of Epilepsy Ognen A. C. Petroff and Dennis D. Spencer Department of Neurology, Yale University School of Medicine, P.O. Box 208018, New Haven, CT 06520-8018, USA

12.1 Introduction 12.2 Part I: MRS Studies of the Role of Astroglial Metabolism in Temporal Lobe Epilepsy 12.2.1 Glutamate Release is Increased and Clearance is Slowed in the Epileptogenic Zone 12.2.2 Glutamate–Glutamine Cycle 12.2.3 Cortical Glutamate Away From the Seizure Focus is Increased in Epilepsy Patients 12.2.4 Cellular Glutamate is Increased in the Epileptogenic Zone 12.2.5 Measuring the Glutamate–Glutamine Cycle in the Epileptogenic Hippocampus 12.2.6 The Glutamate–Glutamine Cycle to TCA Cycle Ratios are Markedly Reduced in the Human Epileptogenic Hippocampus with Severe Neuron Loss and Glial Proliferation 12.2.7 Intracellular Glutamine Content is Low in the Epileptogenic Hippocampus Despite a Major Increase in Glial Density 12.2.8 Proposed Metabolic Model of MTLE with Hippocampal Sclerosis 12.3 Part II: MRS Studies of the Role of GABA in Epilepsy Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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224 225 226 227

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Brain Energetics and Neuronal Activity 12.3.1 Intracellular GABA Levels Modulate the Release and Re-uptake of Extracellular GABA 12.3.2 Cellular GABA and Cortical Excitability 12.3.3 Low Cortical GABA Content is Associated with Low Rates of GABA Synthesis 12.3.4 Inhibition of the GABA–Glutamine or GABA–Glutamate Cycle Results in Seizures 12.4 Conclusion: Alteration of Metabolism in Localization Related Epilepsies and Signiﬁcance for Epileptogenicity

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12.1. INTRODUCTION One out of every 20 Americans will suffer one or more seizures in their lifetime. The application of MRI and MRS to identify localization-related epilepsies has made a major impact on the successful treatment of the disease by surgery. Magnetic resonance methods have been in clinical use for a number of years and permit serial, noninvasive measurements of certain cerebral metabolites without discomfort or known hazards. Several recent reviews examined the value of magnetic resonance imaging (MRI) and spectroscopy (MRS) in the evaluation of the patient with epilepsy (Duncan, 1997; Cascino, 2001; Jackson and Van Paesschen, 2002; Cendes et al., 2002; Hetherington et al., 2002a). N -Acetyl aspartate levels and the ratios of N -acetyl aspartate to creatine (NAA/Cr) are often decreased in the epileptogenic temporal lobe. The changes are usually widespread, can affect the temporal lobe contralateral to the epileptogenic hippocampus and appear to correlate with regions of interictal hypometabolism measured using positron emission tomography. Below normal N -acetyl aspartate or NAA/Cr ratios can provide evidence of temporal lobe abnormalities in epilepsy patients who show no abnormality with MRI. Below normal N -acetyl aspartate in the temporal lobe contralateral to the epileptogenic one removed by surgery can herald a poor outcome. Despite these advances there remains a signiﬁcant fraction of patients with these epilepsy syndromes who do not respond well to surgery or conventional drug treatment. The ability to diagnose patients who fall into this category has the potential of improving the quality of their medical care through both avoiding unnecessary surgery and targeting appropriate drug treatment. Furthermore better mechanistic understanding of the etiology of the disease may both further improve the ability to localize surgically treatable epilepsy, and potentially allow early intervention to prevent the progression of the disease to where surgery is required. In order to address this challenge, we have focused on applying MRS methods for studying brain glutamate and GABA metabolism in epilepsy. Glutamate is the primary excitatory and GABA the primary inhibitory neurotransmitters. Alterations in both glutamatergic and GABAergic systems have been strongly implicated in epileptic syndromes. Continuing MRS development has resulted in techniques for the measurement of cerebral GABA and glutamate in humans, as well as the energetics and neurotransmitter ﬂuxes associated with these compounds. The key linkage between neurotransmission and metabolism exploited by these methods is the role of the astroglia in recycling glutamate and GABA released by the neurons into the synaptic cleft. Once taken up by the astroglia the glutamate or GABA is converted to glutamine. Glutamine is then transported from the glia and taken up by the neuron for conversion to glutamate in glutamatergic neurons and GABA in GABAergic neurons. By using 13 C-labeled precursors the rates of glutamate and GABA neuronal release and recycling may be obtained through analysis of 13 C MRS time courses of glutamate, glutamine, and GABA labeling.

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In this chapter, we describe some of the insights gained into the etiology and pathogenesis of epilepsy through the study of glutamate neurotransmitter cycling and neuroenergetics. The chapter is divided into two parts. The ﬁrst section describes the results of studies on the role of impaired astroglial metabolism in temporal lobe epilepsy. These studies were motivated by the ﬁnding that glutamate clearance is impaired in the damaged epileptogenic hippocampus, resulting in elevated extracellular glutamate even under interictal conditions of low activity (Petroff et al., 2002a). In addition to being excitotoxic, elevated extracellular glutamate leads to neuronal hyperexcitability and potentially increased susceptibility to seizure. The MRS studies have shown that the rate of the glutamate–glutamine cycle is greatly reduced in the sclerotic hippocampus, and that cellular glutamate is elevated (Petroff et al., 2002b). Both of these metabolic changes may be major contributors to the epileptogenic condition. At the end of the section, we propose a model which links the elevated extracellular glutamate measured interictally to a defect in astroglial glutamine synthetase. In Section 12.3 we describe the results of work examining the role of GABA metabolism in epilepsy and the regulation of cortical excitability. These studies stem from advances in MRS technology which have allowed the measurement of cellular GABA and its major metabolites noninvasively in the brain of healthy human subjects (Rothman et al., 1993, 1997; Hyder et al., 1999), and assess how it is altered in epilepsy and its role in the treatment of epilepsy. These studies have found low occipital lobe GABA concentrations in patients with poorly controlled partial seizures, mainly temporal lobe epilepsy (TLE). Raising cellular GABA levels with GABAergic drugs correlates with improvement of seizure control in partial epilepsy patients (Petroff et al., 1996, 1998, 1999a). In addition, below normal cellular GABA levels are found in patients with idiopathic generalized epilepsy (Petroff et al., 2001). These results support a signiﬁcant role for GABA metabolism, as expressed through GABA concentration, in the hyperexcitability associated with the epileptic condition. Consistent with this hypothesis we have shown an association between increasing excitability in the visual cortex and decreasing GABA concentrations induced by light deprivation in healthy human subjects (Boroojerdi et al., 2000a,b).

12.2. PART I: MRS STUDIES OF THE ROLE OF ASTROGLIAL METABOLISM IN TEMPORAL LOBE EPILEPSY Temporal lobe epilepsy (TLE) is a microcosm of all localization-related epilepsies (Spencer, 2002a; de Lanerolle et al., 2003). It has multiple underlying mechanisms, anatomical and molecular substrates, and presentations. Mesial temporal lobe epilepsy (MTLE) is the most common refractory partial epilepsy, and also the one most often treated surgically, because medical treatment fails in 75 % of cases. The combination of neuronal loss of at least 30 % (often greater) in the hippocampus and gliosis is called hippocampal sclerosis (HS), which is the structural correlate that underlies temporal lobe seizures of mesial temporal electrical origin, and together these features deﬁne MTLE. Neoplasms account for 10–15 % of TLE, and are commonly glial in origin. The tumors may invade the hippocampus. Mass-associated TLE (MaTLE) usually refers to non-hippocampal, most often neocortical tumors, that have not invaded the epileptogenic hippocampus. Developmental abnormalities account for another 20 % of the pathological ﬁndings in uncontrolled TLE. Developmental abnormalities are associated with mesial temporal sclerosis in 15–25 % of cases (so-called dual pathology). Conversely, 10–15 % of patients with hippocampal atrophy also have evidence of cortical developmental abnormalities. Finally, trauma accounts for 5–10 % of TLE. An altered neuron to glia ratio with decreased numbers of neurons and increase numbers of astrocytes characterizes most of these pathological states. Astroglial uptake of glutamate plays a key role in maintaining the low extracellular levels needed for proper receptor-mediated functions. Studies of glutamatergic synapses have shown them to be closely surrounded by glial end processes possessing high densities of glutamate transporters (Danbolt, 2001).

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Glutamate transporters are sodium dependent and electrogenic and have an afﬁnity, Km , of 1–3 µM, which is in the range of normal estimated extracellular glutamate concentrations (Bergles et al., 1999; Dzubay and Jahr, 1999). The physiological importance of astroglial glutamate transport was demonstrated in studies in which antisense oligonucleotides directed against the astrocytic glutamate and aspartate transporters GLT1 or GLAST in vivo result in elevated extracellular glutamate in vivo and excitotoxicity (Rothstein et al., 1996). The large majority of cortical glutamate uptake after release is astroglial and tightly coupled to the glial sodium–potassium ATPase and therefore glial energy metabolism (Bergles and Jahr, 1997; Danbolt, 2001). Under severely depolarizing conditions of elevated extracellular potassium and glutamate, which can occur during a seizure, glutamate transporters reverse catastrophically releasing glutamate and aspartate (Rossi et al., 2000; During and Spencer, 1993; Wilson et al., 1996). Compared with controls obtained at autopsy, the resected hippocampus from patients with MTLE and hippocampal sclerosis (decreased neuron densities) demonstrated altered glutamate transporter expression including increased EAAT3 on remaining granule cells and pyramidal neurons, decreased glial EAAT2 (GLT-1) in the hilus and CA1 stratum radiatum associated with neuron loss, and increased glial EAAT1 (GLAST) in CA2/3 stratum radiatum (Mathern et al., 1999). Under normal conditions, GLAST and GLT1 are located on astrocytic membranes. Glia surrounding granular and pyramidal neurons continued to express GLT-1. Glial GLAST expression was increased in 73 % of patients. These ﬁndings, combined with the ﬁnding of increased extracellular glutamate (discussed below), suggest that the increased expression of glutamate transporters were not maintaining effectively extracellular glutamate homeostasis. An increase in glial intracellular sodium, hydrogen ion (acidity) or glutamate content would contribute to glutamate transporter dysfunction by making glutamate uptake energetically more expensive. Under such conditions, failure of extracellular potassium buffering resulting in glial depolarization could be expected to result in reversal of GLAST and a catastrophic release of glutamate.

12.2.1. Glutamate Release is Increased and Clearance is Slowed in the Epileptogenic Zone Dialysate glutamate was reported elevated in spiking neocortex, increasing during seizures (Sherwin, 1999; Wilson et al., 1996). Increasing extracellular glutamate is associated with initiation of seizure discharges in human hippocampus measured in vivo by simultaneous depth electrode electroencephalography and microdialysis. The release of glutamate by the hippocampus contralateral to the seizure focus is less in both amplitude and duration (Figure 12.1). What is unclear is whether the excess glutamate was released by a vesicular (glutamatergic neurons) or nonvesicular (primarily glia) mechanisms. Post-ictal glutamate re-uptake is threefold slower in the epileptogenic hippocampus than the contralateral one. This suggests that glutamate uptake mechanisms, i.e., glutamate transporter function, are slowed or deﬁcient. This ﬁnding is surprising because of severe glutamatergic neuron loss and extensive glial proliferation present in mesial temporal sclerosis resulting in a tenfold increase in the number of glia per neuron (Luby et al., 1995; Spencer et al., 1999, de Lanerolle et al., 2003). Despite the greater density of glia in the atrophic hippocampus, the glia are less effective in clearing extracellular glutamate. Partial dysfunction of the glia slowing the glutamate–glutamine cycle could account for the ﬁndings. Extracellular glutamate levels, measured in the interictal period more than 5 h after the last electrographic seizure, were elevated nearly ﬁvefold in the epileptogenic hippocampus (Cavus et al., 2002). Notably, while the basal extracellular glutamate levels in the non-epileptogenic hippocampi were within known normal limits (less than 5 µM), the levels in the epileptogenic ones were often very elevated, potentially within the neurotoxic range. Paradoxically, interictal, microdialysis glutamate levels were more elevated in those patients with a greater loss of hippocampal neurons. Neuron loss accounted for 70 % of the variance in extracellular glutamate. Raised extracellular glutamate hours into the interictal state suggest a defect in glial glutamate clearance rather than enhanced glutamate release.

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Extracellular glutamate

700% 600% Epileptogenic hippocampus

500% 400% 300%

Contralateral

200% 100% 0% −10

−5

0

5

10

15

20

Time relative to seizure onset (minutes)

Figure 12.1. Microdialysis measurements from paired Spencer electrodes/catheters placed in both hippocampi of patients with MTLE. During a spontaneous seizure, the extracellular glutamate concentrations of the epileptogenic hippocampus increase more than in the contralateral one. Glutamate clearance after the seizure is threefold slower. The ﬁgure is drawn from data published by During and Spencer (1993).

Human glioma, often presenting with seizures, release large amounts of glutamate, raising extracellular glutamate to excitotoxic levels (Ye and Sontheimer, 1999). This mechanism can account for the high extracellular glutamate when epileptogenic glial tumors invade the hippocampus. Unlike normal astrocytes, glial tumors actively take up glutamine and release glutamate. Proliferating glia in the kainic acid model of MTLE also release glutamate into the extracellular space (Stein-Behrens et al., 1994; Yusim et al., 2000). Increased glial glutamate release is attributed to continued activity of the glutamate–cystine antiporter coupled to failure of GLAST and GLT-1. Decreased glial glucose metabolism, associated with decreased glucose transporter (GLUT1) expression and resulting decreased glial energy metabolism, appears to be responsible for decreased glutamate uptake. Glial energy failure would increase intracellular phosphate levels thereby stimulating PAG activity. The enhanced conversion of glutamine to glutamate would raise glial glutamate content.

12.2.2. Glutamate–Glutamine Cycle Astroglia replete neuronal glutamate through the glutamate–glutamine cycle (Figure 12.2). Neurons lack the enzymes required for de novo synthesis of glutamate, and therefore depend on astroglia to provide substrates for the synthesis of glutamate lost during neurotransmission (Hertz et al., 1999). The complete pathway is called the glutamate–glutamine cycle. Studies in the rat cerebral cortex have shown that the rate of the glutamate–glutamine cycle is coupled in a close to 1:1 ratio to neuronal (primarily glutamatergic) glucose oxidation above the rate measured with an isoelectric electroencephalogram (EEG) (Sibson et al., 1998a,b). There is a highly signiﬁcant association between electrical activity measured using EEG, the rate of glucose consumption, and glutamate–glutamine cycling. In the normal adult brain, the glutamate concentration of gray matter primarily reﬂects the glutamate concentration in glutamatergic neurons, whose glutamate content is far greater than nonglutamatergic neurons or glia (Ottersen et al., 1992). Neuronal glutamate is lost during glutamate transmitter release and is taken up by glia where it is recycled by glutamine synthetase (Danbolt, 2001). With increased excitatory activity, the rate of neuronal glutamate loss would be greater. Glutamate lost from the neuron is replaced through phosphate-activated glutaminase (PAG) acting upon glutamine synthesized in the glia (Fonnum, 1993; Conti and Minelli, 1994). Inhibition of PAG or blockade of glutamine transporters slows

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Brain Energetics and Neuronal Activity Model of neuron-glia cycle (concentrations compiled from the human & rat literature) Capillary

Astrocyte

ECF

GLN 5–6 mM

GLN 550 µM

Glutamate neuron Components of glutamate-glutamine cycle

GLN 650 µM

GLN <1 mM

Glutamate-glutamine cycling NH3 <100 µM

Leucine 150 µM

Glucose 5 mM

2. Glutamate uptake - primarily glial transporters

NH3 < 50 µM GLT <1 mM

GLT < 5 µM

1. Glutamate release by neuron - neurotransmission

3. Glutamine synthesis - glial glutamine synthetase GLN 10–15 mM

4. Glutamine release by glia - transporter mediated

TCA cycle

TCA cycle

5. Glutamine uptake by neuron - transporter mediated

Pyruvate carboxylase

Pyruvate dehydrogenase

6. Glutamate synthesis - phosphate activated glutaminase

Pyruvate Pyruvate

Figure 12.2. Schematic diagram of the glutamate–glutamine cycle between neurons and glia. The diagram is adapted from Petroff et al. (2002a).

the glutamate–glutamine cycle and decreases neuronal glutamate and GABA (Rae et al., 2003; Chaudry et al., 2002).

12.2.3. Cortical Glutamate Away From the Seizure Focus is Increased in Epilepsy Patients Extensive studies in animals have shown that changes in glutamate release and metabolism may play an important role in the origin and spread of seizure activity (Bradford, 1995; Sherwin and van Gelder, 1986). Glutamate metabolism is coupled closely to mitochondrial respiration and ATP synthesis. Neurotransmitter glutamate release from synaptosomes is drastically reduced if either ATP or cytosolic glutamate concentrations are depleted, which is consistent with a critical role for cytosolic glutamate metabolism for maintaining the vesicular pool. As a result of the relationship between cytosolic and vesicular glutamate concentrations, an increase in cytosolic glutamate appears to enhance vesicular glutamate release. Glia efﬁciently remove glutamate released by neurons and help to terminate the action of released glutamate. In vitro and in vivo studies indicate that a major portion of glutamate transported into glia is converted to glutamine (neutral metabolite) and returned to neurons for the resynthesis of glutamate. Disturbances of the glutamate–glutamine cycle, responsible for the increased intracellular glutamate levels and impaired glutamate clearance could be an important metabolic change associated with epileptogenesis. Neocortical intracellular glutamate levels are increased in the epileptic human brain. Biopsies of human temporal lobe cortex ipsilateral to the epileptogenic hippocampus show that glutamate is elevated by 2.3 µmol/g in spiking cortex compared with adjacent non-spiking cortex (Sherwin, 1999). Biopsies of epileptic, but not necessarily epileptogenic, neocortex ipsilateral to the seizure focus show an increase in intracellular glutamate content of 0.4 to 2.1 µmol/g compared to tissue resected from the margin of tumors of patients without seizures (reviewed in Petroff et al., 1995). Proton MRS has been used to measure intracellular glutamate levels (Petroff et al., 1995, 1999a, 2000, 2002c). Occipital lobe glutamate levels of localization-related epilepsy patients with frequent seizures treated with carbamazepine or phenytoin are higher than those of epilepsy-free, drug-free volunteers (Figure 12.3). Mean glutamate content remote from the seizure focus is 0.7 mM greater in our patients with refractory complex partial seizures than control subjects. Glutamate plus glutamine (GLX) levels

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Occipital glutamate (mM)

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8 NO epilepsy

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Ipsilateral

Figure 12.3. Graph on the left shows that mean intracellular glutamate levels (± standard error) measured using proton-MRS from the occipital lobe of nine patients with refractory, complex partial seizures treated with carbamazepine or phenytoin and ten drug-free, epilepsy-free volunteers. Intracellular glutamate levels remote from the seizure focus are increased, potentially contributing to the spread of seizure activity. The graph is drawn from data published by Petroff et al. (2000). Graph on the right shows the mean ratios of glutamate plus glutamine (GLX) to creatine plus phosphocreatine (CR) measured by in vivo proton MRS of the human temporal lobe. The graph is drawn from data published by Savic et al. (2000).

Intracellular glutamate (mM)

8

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5 Epileptogenic hippocampus

Autopsy hippocampus

Figure 12.4. Mean intracellular glutamate levels (± standard error) of portion of the hippocampus removed at surgery for TLE compared to values reported for histologically normal hippocampi obtained at autopsy. Neuron loss is measured in an adjacent slice of the pes. Intracellular glutamate concentrations are elevated in the epileptogenic human hippocampus removed by surgery. Graph is drawn from data that includes values published by Petroff et al. (2002a).

appear to be increased in the temporal lobe ipsilateral to the seizure onset (Woermann et al., 1999; Savic et al., 2000). The increased neocortical glutamate levels ipsilateral to the epileptogenic temporal lobe suggest that increased intracellular glutamate content could contribute to the hyperexcitability of the epileptic network facilitating spread of seizure activity to brain regions outside of the ictal onset zone. Imaging glutamate may become very useful in mapping epileptic networks (Spencer, 2002b).

12.2.4. Cellular Glutamate is Increased in the Epileptogenic Zone Intracellular glutamate concentrations are elevated in the epileptogenic human hippocampus (Figure 12.4). The elevation is even more striking when hippocampal glutamate content is corrected for 60 % neuron

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loss (Petroff et al., 2002a). Compared with the autopsy series mean, hippocampal glutamate is elevated in MTLE patients. Elevated glutamate content is present in hippocampi with a normal clinical MRI appearance. Even higher levels are present in almost half the hippocampi with the greatest neuron loss. Intracellular glutamate concentrations must be exceedingly high in the remaining glutamatergic neurons or above normal, intracellular, glutamate concentrations must be present in the remaining non-glutamatergic neurons or glia. Brain glutamate levels should decrease in proportion to neuronal loss or simpliﬁcation (loss of neuronal volume through shrinkage of dendrite, synapses, and other processes). The neuron loss, particularly of large glutamatergic neurons, and glial proliferation should decrease glutamate concentrations in the sclerotic hippocampus. Intracellular glutamate and glutamine content are the same in biopsies of epileptogenic hippocampi with the least and most severe neuron loss. Hippocampal neuron loss appeared to have little impact on the cellular content of glutamate (R2 < 0.01) or glutamine (R2 < 0.01). Our data were quite remarkable in that they failed to show any signiﬁcant relationship between the degree of neuronal loss and the cellular content of glutamate (a major neuronal metabolite) and glutamine (a major glial metabolite). Rank-order regression shows that less than 2 % of the variability in cellular metabolite content is accounted for by the fourfold difference in the degree of neuron loss. Our ﬁndings suggest that there appears to be a relative increase in cellular glutamate content in the epileptogenic human hippocampus. Under normal conditions, increased tissue glutamate would reﬂect increased intracellular levels in the glutamatergic neurons. However, one third of the epileptogenic HS hippocampus with more than 60 % neuron loss had the highest glutamate concentrations. The other subpopulation with clearly above normal glutamate content was those hippocampi with the least neuron loss (Ma-TLE and PTLE groups, see de Lanerolle et al., 2003). The ﬁndings raise two possibilities. Either the remaining neurons, mainly ‘GABAergic’, have extremely high intracellular glutamate content or a subpopulation of glial cells must have high glutamate content. Cell culture studies suggest that glial precursor cells have high intracellular glutamate content (Urenjak et al., 1993; Bhakoo and Pearce, 2000). Above normal intracellular glutamate concentrations could contribute to the above normal release of glutamate measured in the epileptogenic human hippocampus during spontaneous seizures. The high glutamate content would be expected to contribute to the epileptic state by increasing network excitability and promoting excitotoxicity.

12.2.5. Measuring the Glutamate–Glutamine Cycle in the Epileptogenic Hippocampus In order to determine whether glutamate–glutamine cycling is impaired in MTLE we have developed a strategy for determining the ratio of the glutamate–glutamine cycle (Vcycle ) to the rate of the total glutamate synthesis rate, TCA cycle (VTCA ) as well as the total glutamine synthesis (VGS ) from steady state 13 C labeling patterns in glutamate and glutamine (Figure 12.5). The strategy takes advantage of the label from 2-13 C -glucose being incorporated into the internal positions of glutamate and glutamine only through the glia (Sibson et al., 2001; Petroff et al., 2002b). Label from 2-13 C -glucose, which enters the TCA cycle through pyruvate dehydrogenase (PDH), is incorporated into 5-13 C -glutamate and 1-13 C-glutamate. It does not label the internal positions of glutamate or glutamine. In contrast, 13 C label entering the TCA cycle from the anaplerotic pathway through pyruvate carboxylase will label glial glutamine C2 and C3 initially because both pyruvate carboxylase (PC) and glutamine synthetase (GS) are found exclusively in glia. Subsequently labeled glutamine will be taken up by the neurons to replenish released glutamate by the glutamate–glutamine cycle. The labeling in neuronal C3-glutamate is diluted relative to the precursor C3glutamine due to unlabeled carbons entering through the neuronal TCA cycle (Vunlabeled ). From the ratio of C3-glutamine to C3-glutamate labeling at isotopic steady state the ratio of the rate of glutamate–glutamine cycle to neuronal TCA cycle (Vcycle /VTCA ) may be calculated using the equations given below.

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Astrocyte

5-13C-glutamine

5-13C-glutamine

Vcycle

PAG VPAG

GS

5-13C-glutamate

VGS

5-13C-glutamate glial TCA

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Pyruvate dehydrogenase

Pyruvate dehydrogenase Glycolysis

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PAG

VPAG

3-13C-glutamate

3-13C-glutamine

Vcycle

GS

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3-13C-glutamate

Vunlabeled Vanaplerosis VTCA

Pyruvate carboxylase

Glycolysis

2-13C-glucose

Glycolysis

2-13C-glucose

Figure 12.5. Schematic diagram of 2-13 C-glucose metabolism adapted from Petroff et al. (2002b).

At isotopic steady state the label inﬂows and efﬂuxes from the glutamate and glutamine pools must be equal and the isotopic enrichment of 3-13 C-glutamate is determined by the following isotopic balance equation: d[3-13 C-glutamate]/dt = 0 = GN3Vcycle + 0.5(GT3 + GT4)VTCA − GT3(Vcycle + VTCA )

(12.1)

where GN3 is the isotopic enrichment of the C3 position of glutamine and GT3 the isotopic enrichment of C3-glutamate. Rearrangement of the above expression and solving for the Vcycle /VTCA ratio yields Vcycle /VTCA = 0.5(GT3 − GT4)/(GN3 − GT3)

(12.2)

Because tissue glutamate concentrations primarily reﬂect the glutamate content of glutamatergic neurons, the variable VTCA monitors the mitochondrial activity of the glutamatergic neuron. We have validated this strategy of measuring the glutamate cycle/TCA cycle ratio in the rat cerebral cortex under chloralose anesthesia by comparison of the rates calculated from the steady state 2-13 Cglucose experiment with those calculated from dynamic 1-13 C -glucose, 2-13 C - glucose, and 5-13 C -glucose studies (Figure 12.6) (Sibson et al., 1998a, 2001). Similar results have been obtained using 14 C-labeled carbon dioxide, which also incorporates label through pyruvate carboxylase (Lieth et al., 2001). Although the TCA cycle rate is twofold faster in the rat than in humans, the glutamate cycle/TCA cycle ratio is similar in both (Shen et al., 1999; Kanamatsu and Tsukada, 1999). The glutamate cycle/TCA cycle ratio is clearly decreased by surgical-grade anesthesia. However, the effects are minor compared with the major decrease with the isoelectric EEG of pentobarbital coma. Changes in rate of glutamate turnover and thus the glutamate–glutamine cycle are proportional to EEG power and changes in the neuronal spiking frequency (Hyder et al., 2001; Smith et al., 2002). The steady-state measurement has signiﬁcant advantages clinically. It allows the stable isotope tracer to be administered under more ﬂexibly controlled conditions before the MRS measurements, thereby allowing more magnet time for spectral acquisition. The signal-to-noise ratio is improved or voxel size reduced. Administering the 13 C or 15 N tracer before the MRS measurements facilitates infusion under a greater variety of conditions often with better control of the experimental paradigms and with greater subject comfort.

Brain Energetics and Neuronal Activity 0.6 Awake human

0.5

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0.4 0.3

Glutamate-glutamine cycle to TCA cycle ratio

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Pentobarbital Morphine Chloralose 30 mg/kg 50 mg/kg 80 mg/kg

0.2 0.4

1.0 1.2 0.6 0.8 Tricarboxylic acid (TCA) cycle rate

1.4

0.7 0.6

Same animals used in concurrent analysis

0.5 0.4 0.3 0.2 0.1 0.0 Dynamic model Steady state Infusion of 2-13C-glucose

Figure 12.6. The graph on the left shows the ratio of the glutamate–glutamine cycle rate normalized to the TCA cycle rate plotted as a function of the rate of the TCA cycle under a variety of levels of brain activity, e.g., depth of anesthesia. The data is derived from Sibson et al. (1998a), Hyder et al. (2001), Shen et al. (1999), and Kanamatsu and Tsukada (1999). Graph on the right shows that the ratios of the glutamate–glutamine cycle rate to TCA cycle rate, measured using serial spectra, are the same as those measured in a single spectrum, collected after steady state is achieved. Serial spectra, collected during the dynamic phase of the experiment, and the steady-state spectrum at the end are obtained from the same animal. The graph is drawn from data published by Sibson et al. (2001).

Glutamate-glutamine cycle to tricarboxylic acid cycle ratio

0.5 0.4 0.3 0.2 0.1 0.0 Epileptogenic hippocampus

Normal cerebrum

Figure 12.7. The ratio of the glutamate–glutamine cycle rate to TCA cycle rate (± standard error) of a portion of the hippocampus removed at surgery for TLE. The glutamate–glutamine cycle/TCA cycle ratio is compromised severely in almost all patients. Using the ratio normalizes for variations in the cerebral metabolic rate. The greatly reduced glutamate–glutamine cycle rate accounts for slow glutamate clearance from the synapse, increased interictal extracellular glutamate concentrations, and contributes to excitotoxicity. The graph is drawn from data that includes values published by Petroff et al. (2002b).

12.2.6. The Glutamate–Glutamine Cycle to TCA Cycle Ratios are Markedly Reduced in the Human Epileptogenic Hippocampus with Severe Neuron Loss and Glial Proliferation Few reports address the effects of neuron loss and gliosis on the metabolite content of the epileptogenic human hippocampus. We showed that the glutamate–glutamine cycle/TCA cycle ratio is severely compromised in almost all patients (Figure 12.7) (Petroff et al., 2002b). No associations with antiepileptic drugs

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in use at the time of surgery, duration of the epilepsy, gender, or age were seen. There were no significant associations between glutamate–glutamine cycle to the neuronal TCA cycle ratio and hippocampal glutamate or glutamine content. Measurements of the glutamate–glutamine cycle to the neuronal TCA cycle ratio in the normal human hippocampus are not available. The closest comparisons to our results are recent measurements using in vivo 13 C MRS in the human occipital–parietal lobe and the awake and lightly anesthetized rat forebrain (Shen et al., 1999; Kanamatsu and Tsukada, 1999). Under low light, unstimulated conditions, values range between 0.4 to 0.5. The relative rate of the glutamate–glutamine cycle to glutamate synthesis is decreased in epileptic hippocampi that show sclerosis. The association with histopathology is striking. Neuron–glia cycling is lowest in epileptic hippocampi with signiﬁcant loss of neurons and glial proliferation. Because this ratio is calculated from the relative labeling of the glutamate (primarily neuronal) and glutamine (synthesized in glia) pools, as opposed to absolute ﬂux rates, the low ratio of the glutamate–glutamine cycle to the neuronal TCA cycle is not simply due to reduced cellular density or generalized hypometabolism. If the remaining neurons and glia were functioning normally the ratio of glutamate–glutamine cycle to the neuronal TCA cycle would be independent of neuronal loss.

12.2.7. Intracellular Glutamine Content is Low in the Epileptogenic Hippocampus Despite a Major Increase in Glial Density Hippocampal glutamine concentrations are surprisingly low in the epileptogenic human hippocampus (Figure 12.8) (Petroff et al., 2002a). Our cellular glutamine values appear to be low compared to the cellular glutamine concentration of the hippocampus obtained at autopsy from nondemented subjects (Tarbit et al., 1980). Brain tissue glutamine content primarily reﬂects glial concentrations (Ottersen et al., 1992; Kvamme et al., 2000). The lack of association between cellular glutamine and pathology, reﬂecting the degree of glial proliferation and neuron loss, suggests that glial glutamine levels are low in most cases of hippocampal sclerosis. When corrected for glial density, intracellular glutamine levels are exceedingly low (1.5 mM, SD 0.7). The lowest values are found in epileptogenic glioma involving the hippocampus with mean intracellular glutamine levels (0.7 mM) similar to values measured in the extracellular space by microdialysis.

Intracellular glutamine (mM)

5 4 3 2 1 0 Epileptogenic hippocampus

Autopsy hippocampus

Figure 12.8. Mean intracellular glutamine levels (± standard error) of a portion of the hippocampus removed at surgery for TLE. Glial proliferation is measured in an adjacent slice of the pes. Decreased expression of glutamine synthetase, measure by quantitative electron microscopy of the resected hippocampus, can account for the low glutamine content, despite the large increase in glial density. The graph is drawn from data that includes values published by Petroff et al. (2002a).

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The immunohistochemical distribution of GS was similar in autopsy and non-MTLE hippocampi (Eid et al., 2002). In hippocampi without reactive glial proliferation, GS was strongly expressed in glial cells throughout all subﬁelds. In contrast, in the epileptogenic and sclerotic hippocampus, a profound loss of GS-immunoreactivity was apparent, particularly in areas of peak neuronal loss and glial proliferation, such as the CA1 and CA3 regions. Western blot analysis showed that GS was down-regulated by 40 % in the MTLE hippocampus compared to surgically resected non-MTLE hippocampi. No GS-immunoreactivity was seen in neurons. The deﬁciency in GS in the sclerotic regions of the MTLE hippocampus would be a sufﬁcient mechanism for the increase in extracellular glutamate and decrease in intracellular glutamine. Glial glutamate content should be increased. In vitro experiments with organotypical hippocampal cultures have demonstrated that blocking of GS by the competitive inhibitor methionine sulfoximine (MSO) causes depletion of glutamine and accumulation of glutamate in astrocytes (Laake et al., 1995). Acute administration of inhibitors of glutamine synthesis, e.g. methionine sulfoximine, results in convulsions with a lag of several hours (Sellinger et al., 1986). In the ferric chloride model of focal epilepsy, glutamine synthetase activity is reduced in the seizure focus (Tiffany-Castiglioni et al., 1989). Inhibition of glutamine synthesis would be expected to cause slow glutamate–glutamine cycling, to decrease tissue glutamine content and release, thereby, to raise glial glutamate content and efﬂux. During glial depolarization, enhanced glutamate efﬂux would be expected to foster spread of the seizure. Low rates of glutamate–glutamine cycling resulting from dysfunction of glutamine synthesis could contribute to the epileptic state even under conditions of severe neuron loss and gliosis.

12.2.8. Proposed Metabolic Model of MTLE with Hippocampal Sclerosis Our proposed model (Figure 12.9) is based on the anatomical observation that glia to neuron ratios are increased tenfold in hippocampal sclerosis with MTLE. Neuron density is severely reduced and glial density is more than doubled. The glutamate–glutamine cycle rate is very low; reduced by a factor of four or more. Interictal extracellular glutamate concentrations are increased by a factor of three to ﬁve. Intracellular glutamate concentrations are unchanged despite the neuron loss. Intracellular glutamine concentrations are low despite an over twofold increase in glial density. Glial dysfunction would account for all these observations. Increased glutamate release from neurons would be unlikely because of the profound neuron loss. Increased neuronal release of glutamate should increase rather than decrease the rate of Astrocyte

GLN 1– 2 mM

ECF

GLN 600 µM

Glutamate neuron

GLN <1 mM

Glutamate-glutamine cycling

GLT 3 – 5 mM

GLT 15 µM

GLT 10 –15 mM

Figure 12.9. Schematic diagram of our hypothesis that dysfunction of the glutamate–glutamine cycle helps maintain the epileptogenic state in MTLE. Increased intracellular glutamate and decreased glial glutamate clearance maintain increased extracellular glutamate concentrations that promote spontaneous seizures.

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glutamate–glutamine cycling. Furthermore, GS expression is severely reduced in hippocampal sclerosis; virtually absent from astrocytes in the most gliotic regions. Loss of this critical enzyme could account for many of our observations. Glial intracellular glutamine content should decrease. Glial glutamate content should increase. The neuron–glia, glutamate–glutamine cycling would slow. The increase in glial glutamate would make glial glutamate uptake energetically more costly and prolong clearance of glutamate from the extracellular ﬂuid (ECF). Interictal extracellular glutamate would be chronically elevated. Glial depolarization would increase extracellular glutamate further, perhaps by reversal of GLAST or GLT-1. Hippocampal GLAST expression is increased in the majority of patients.

12.3. PART II: MRS STUDIES OF THE ROLE OF GABA IN EPILEPSY GABA plays an important role in mammalian epilepsy. GABA is the major inhibitory neurotransmitter in the mammalian brain, including humans. Reviews by several investigators have stressed the role of GABA in epilepsy and emphasized the developmental aspects of GABA physiology, with implications for childhood epilepsy (van den Pol et al., 1998; Powell et al., 2003; Berkovic and Scheffer, 1999; Prasad et al., 1999). Animal models using advanced molecular genetic techniques such as knockout mutations have identiﬁed the role of genes affecting ion channels, the synapse (including release and uptake of neurotransmitters) synaptic receptors and the neural network that result in epilepsy. Many of these mutations result in alterations of GABA and glutamate physiology. Mutation of a nonspeciﬁc alkaline phosphatase results in markedly reduced brain GABA and intractable seizures (Waymire et al., 1995). In addition, rare genetic disorders of GABA metabolism have been identiﬁed in a few hundred children (Jakobs et al., 1993). These disorders include pyridoxine-dependent epilepsies, GABA-transaminase deﬁciency and succinic-semialdehyde-dehydrogenase deﬁciency (Battaglioli et al., 2000; Gibson et al., 1998; MedinaKauwe et al., 1999). Subjects with pyridoxine-dependent epilepsies have been observed to have extremely low CSF GABA levels, elevated glutamate, and low levels of brain GABA (Kurlemann et al., 1992; Kure et al., 1998; Battaglioli et al., 2000). Recent reports have suggested that similar changes in CSF GABA occur in children with infantile spasms (Ohtsuka et al., 2000). Despite the known role of pyridoxine (vitamin B6) as an important cofactor for glutamic acid decarboxylase (GAD), a key regulatory enzyme of GABA metabolism, characterization of the molecular defect in pyridoxine dependent epilepsies has yet to be discovered.

12.3.1. Intracellular GABA Levels Modulate the Release and Re-uptake of Extracellular GABA Changes in cytosolic GABA concentrations alter cellular physiology in a variety of ways. Under normal conditions, the GABA content of GABAergic neurons is far greater than that of non-GABAergic neurons or glia. Neuronal GABA concentrations may inﬂuence both vesicular and non-vesicular GABA release. Vesicular uptake of GABA, thus the vesicular concentration, is dependent on cytosolic GABA concentration (Km = 5 mM) and energy charge (Fyske and Fonnum, 1996). An increase in cytosolic GABA will enhance vesicular GABA release (Gram et al., 1989). Tonic GABA release through reversal of GAT1 is concentration dependent. Release of GABA through transporter reversal has been demonstrated with both physiological and epileptic stimuli (Gaspary et al., 1998; Wu et al., 2001). Tonic release of GABA increases with increasing cytosolic GABA resulting in increased basal extracellular levels. The distribution of GAD67 appears to coincide with the distribution of the neuronal GABA transporter (GAT1), suggesting that cytosolic GABA synthesized by this GAD isoform may have a signiﬁcant paracrine function, perhaps through transporter reversal (Yasumi et al., 1997; Gaspary et al., 1998; Wu et al., 2001). Thus, an increase in cytosolic GABA will enhance both vesicular and nonvesicular GABA release during seizures and slow clearance of GABA from the synaptic cleft thereby prolonging its effects (Gram et al., 1989; Gaspary

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et al., 1998). Dynamic insertion of GABA transporters into the plasma membrane occurs in response to increased extracellular GABA. Increased transporter availability interacting with higher cytosolic GABA would act in a synergistic fashion to magnify GABAergic effects. Conversely, within the epileptogenic and sclerotic human hippocampus GABA content, release, and transporter function appear decreased (During et al., 1995; Patrylo et al., 2001).

12.3.2. Cellular GABA and Cortical Excitability Increased cortical excitability is a hallmark of several human epileptic syndromes. Several lines of evidence support a major role for GABA levels affecting GABA release and by this mechanism having a key role in the regulation of cortical excitability. Studies in cell culture and brain slice have shown directly that increasing cellular GABA levels increases tonic GABA release in response to physiological activation (Gaspary et al., 1998; Wu et al., 2001). Primate models of photosensitive epilepsy have low GABA levels and improve with GABAergic drugs (Menini and Silva-Barrat, 1997; Lloyd et al., 1986). Similarly, AEDs that increase GABA or enhance GABAergic inhibition block the photoparoxysmal response in photosensitivity epilepsies (Kasteleijn-Nolst Trenit´e, 1998; Rimmer et al., 1987). Drug-induced enhancement of GABAergic inhibition reduces abnormal ﬂash-evoked potentials in parallel with an improvement in the associated myoclonus (Cracco and Rossini, 1997). In normal volunteers, 1 h of low ambient lighting (darkness) increases the excitability of the visual cortex (Boroojerdi et al., 2000a,b). Pretreatment with a GABA-A receptor agonist (lorazepam) abolishes the increase in excitability, measured using fMRI and transcranial magnetic stimulation. Serial MRS measurements of the visual cortex show that intracellular GABA content decreases in parallel to the enhanced cortical excitability. Myoclonus without loss of consciousness, characteristic of JME and other myoclonic epilepsy syndromes is attributed to a defect in GABAergic inhibition (Brown et al., 1996; Werhahn et al., 1999). Spontaneous and photosensitive seizures are seen in alcohol and other drug withdrawal states characterized by downregulation of the GABA-A receptors or low occipital GABA levels (Behar et al., 1999). MRS-based measurements show low GABA levels in the visual cortex of patients, consistent with the enhanced cortical excitability observed in JME (Petroff et al., 2001). Intracellular homocarnosine and GABA levels are often below normal in the neocortex of patients with frequent seizures. Low tissue GABA levels are associated with increased cortical excitability that would facilitate the spread of seizure activity from the epileptogenic hippocampus to the neocortex. Studies using MRS have found reduced cellular GABA levels in the human visual cortex in localizationrelated epilepsies (Figure 12.10). Below normal GABA levels in the visual cortex are associated with poor seizure control in refractory localization-related epilepsy syndromes (Petroff and Rothman, 1998; Petroff et al., 1996, 1998, 1999b, 2001). Whether low GABA levels are the cause or the result of frequent seizures is unknown. Both GABA concentrations and synthesis rates increase in most seizure models (Bradford, 1995). It has been proposed that enhanced glutamic acid decarboxylase (GAD67 isoform) mRNA expression, enzyme activity, and GABA levels that occur within hours in animal models of stress, seizures, and other injuries could reﬂect a protective response allowing the brain to heal by producing widespread inhibition (Martin and Tobin, 2000). Therefore, it is surprising that occipital GABA levels are below normal in many patients with poor seizure control (Figure 12.10), particularly in regions presumably remote from the epileptogenic areas (most often temporal and frontal lobes in our series). Low GABA levels, measured in the occipital cortex remote from the onset of the seizure, correlate with epileptiform abnormalities seen in interictal EEG recording (Figure 12.11) (Petroff et al., 1996). Some studies have shown that patients with epileptiform abnormalities on interictal EEG recorded shortly after a seizure are more likely to have another seizure within 2 years than those with normal EEGs (van Donselaar et al., 1992). These interictal epileptiform abnormalities probably reﬂect increased cortical excitability.

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Occipital edited GABA (mM)

1.5 1.4 1.3 1.2 1.1 1.0 Good

Poor Seizure control

Figure 12.10. Mean edited GABA levels (± standard error) measured using proton-MRS from the occipital lobe of 42 patients with localization-related epilepsy treated with carbamazepine or phenytoin. Levels are signiﬁcantly lower in patients with refractory complex partial seizures. In humans, edited GABA is the sum of GABA and homocarnosine. Low cortical GABA and homocarnosine levels facilitate the spread of seizures from the epileptic focus and contribute to the cortical excitability, which characterizes the epileptic network. Data include values published by Petroff et al. (1996, 1999a, 2000).

Cellular edited GABA (mM)

1.2

1.1

1.0

0.9

0.8 Normal EEG

Non-specific abnormality

Epileptiform abnormality

Figure 12.11. Mean edited GABA levels (± standard error) measured using proton-MRS from the occipital lobe of patients with localization related epilepsy. Levels are lowest in patients, whose interictal electroencephalogram (EEG) reveals epileptiform features that correlate with increased cortical excitability and a high risk of recurrent seizures. Measuring interictal edited GABA using proton-MRS may complement the EEG in predicting seizure recurrence. Spectroscopic imaging may deﬁne the epileptic network. The graph is drawn from data published by Petroff et al. (1996).

Similarly, localization of the site of termination of seizures of focal origin to cortical regions other than the onset is associated with a poorer surgical prognosis (Spencer and Spencer, 1996). This observation raises the possibility of additional abnormal epileptogenic cortical regions with impaired seizure-terminating capabilities, i.e., increased cortical excitability. The presence of interictal epileptiform discharges extending beyond the area of resection correlates with poor surgical outcome in patients with extra-hippocampal epilepsy (Bautista et al., 1999). The observations are consistent with the hypothesis that increased cortical excitability beyond the epileptogenic region appears to contribute continued seizure activity. Low cellular

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GABA, measured by MRS in the neocortex away from the seizure focus, reﬂects regions of increased excitability and may contribute to the spread of seizures.

12.3.3. Low Cortical GABA Content is Associated with Low Rates of GABA Synthesis GABAergic neurons are decreased in the human epileptogenic neocortex associated with temporal lobe epilepsy (TLE), low-grade tumors, and cortical malformations (Ribak and Yan, 2000; Haglund et al., 1992; Marco et al., 1996; Spreaﬁco et al., 1998). Loss of GABAergic processes is seen in some cases. Low rates of GABA synthesis are measured in the occipital lobe of patients with refractory, localization-related epilepsy (Figure 12.12). These patients have normal function of the visual cortex and no anatomical lesion or atrophy of the occipital lobe. The net rate of GABA synthesis was measured using serial MR spectra in patients following the inhibition of GABA-transaminase with vigabatrin (Petroff et al., 1999c). GABA is formed from the alphadecarboxylation of glutamate by glutamic acid decarboxylase (GAD) and is metabolized to succinate by the sequential actions of GABA transaminase (GABA-T) and succinic semi-aldehyde-dehydrogenase (SSADH). The activity of GAD is believed to be primarily responsible for regulating the steady-state concentration of GABA (Martin and Tobin, 2000; Martin and Rimvall, 1993). Low intracellular GABA content appears to be associated with low rates of GABA synthesis in human epilepsy.

12.3.4. Inhibition of the GABA–Glutamine or GABA–Glutamate Cycle Results in Seizures The GABA concentration of neocortex primarily reﬂects the fractional volume of GABAergic neurons weighted by the GABA content of those neurons. One explanation for low intracellular GABA levels in poorly controlled epilepsy would be loss of GABA-containing neurons or a decrease in their volume (simpliﬁcation of dendritic and synaptic tree or loss of axonal length and thickness). Low rates of GABA synthesis result from genetic or acquired defects in GAD expression or function (Kash et al., 1997; Battaglioli et al., 2000; Peltola et al., 2000). Mice deﬁcient in GAD develop seizures. Pyridoxine deﬁciency results

Net rate of GABA synthesis (mM/hour)

2.0 1.8 1.6 1.4 1.2 1.0 0.4

0.6

0.8

1.0

1.2

Cortical intracellular GABA (mM)

Figure 12.12. The net rate of GABA synthesis in six patients with refractory, localization-related epilepsy, measured using serial proton-MRS after the ﬁrst dose of a GABA-transaminase inhibitor, vigabatrin. There is an association between cortical GABA content and the net rate of synthesis, suggesting that low rates of synthesis accounts for the low GABA content rather than increased catabolism. The graph is drawn from data published by Petroff et al. (1999c).

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in epilepsy with low GABA levels. Circulating auto-antibodies against GAD occur in some patients with refractory epilepsy. Alternatively, the availability of glutamate may be rate limiting. GABAergic neurons appear to be unable to synthesize glutamate de novo from glucose or lactate. Glutamine, produced by glia, serves as a major precursor of the releasable pool of GABA (Kapetanovic et al., 1993; Waniewski and Martin, 1998; Patel et al., 2001; Rae et al., 2003). Disrupting the GABA–glutamine cycle would be expected to decrease glutamine availability, reducing the glutamate content of GABAergic neurons, thereby decreasing the rate of GABA synthesis. Inhibition of neuronal glutamine uptake or inhibition of neuronal glutaminase appears to result in decreased GABAergic inhibition, before loss of glutamatergic excitation, thereby facilitating seizures. However, disruption of the glutamate–GABA cycle also results in low intracellular GABA content. A signiﬁcant proportion of GABA is synthesized from glutamate transported via EAAT3 glutamate transporters. Inhibiting neuronal glutamate transporters reduced both evoked monosynaptic and miniature IPSCs with no change in the frequency of IPSCs or in the response to exogenously applied GABA, suggesting that GABA synthesis and, consequently, vesicular ﬁlling were reduced (Mathews and Diamond, 2003). Similarly, a knockdown in glutamate transporter expression was associated with a decrease in GABAergic function in mice and seizure activity (Sepkuty et al., 2002). Dysfunction of the glutamate–GABA cycle (glutamatergic neurons express GABA transporters and GABA-transaminase) results in low rates of GABA synthesis and seizures. Further, labeling studies should clarify which mechanism accounts for low GABA content in an individual patient. Occipital–posterior–temporal lobe GABA+/CR ratios are reported to be 30 % lower ipsilateral to the seizure focus (Figure 12.13) (Mueller et al., 2001). The ratios are lowest ipsilateral to the seizure focus in those patients who achieved good seizure control after GABA levels are increased with vigabatrin. Vigabatrin is a GABA-transaminase inhibitor that increases intracellular and extracellular GABA and homocarnosine (Petroff and Rothman 1998, Petroff et al., 1998). This suggests that increased GABA may contribute to the reduction in symptoms. A recent review proposes that human epilepsy is a disorder of large neural networks (Spencer, 2002b). The electrical hyperexcitability associated with seizure activity reverberates within the neural structures of the network to culminate in the eventual expression of seizures. The most extensively described of these epilepsy networks, the medial temporal or limbic network, includes the hippocampi, the amygdala, the

Occipital GABA+ / CR ratio

0.15

0.10

0.05 Ipsilateral

Contralateral

With respect to the seizure focus

Figure 12.13. Proton MRS measurements of the GABA+ to creatine ratio (GABA+/CR) of the occipital lobe ipsilateral and contralateral to the seizure focus in patients with TLE. The ratio is signiﬁcantly less on the ipsilateral side. Low GABA may deﬁne the epileptic network in localization-related epilepsies. The graph is drawn from data published by Mueller et al. (2001).

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entorhinal cortices, lateral temporal neocortices, and extra-temporal components of the medial thalamus and the inferior frontal lobes. The other two networks are less commonly identiﬁed, even in their component parts: the medial occipital/lateral temporal network and the superior parietal/medial frontal network. The electrical hyperexcitability associated with seizure activity reverberates within the neural structures of the network, which operate together and inextricably to culminate in the eventual expression of seizures. Modiﬁcation of network activity by electrical, biochemical, or metabolic inﬂuences in any part of the network will alter seizure expression or its occurrence. The low neocortical GABA levels ipsilateral to the epileptogenic temporal lobe suggest that low cellular GABA could contribute to the hyperexcitability of the epileptic network. Imaging GABA should become very useful in mapping epileptic networks.

12.4. CONCLUSION: ALTERATION OF METABOLISM IN LOCALIZATION RELATED EPILEPSIES AND SIGNIFICANCE FOR EPILEPTOGENICITY The studies reviewed in this chapter have focused on alterations in the metabolism, particularly astroglial neurotransmitter cycling, of glutamate and GABA in localization-related epilepsy. Based on these studies there appears to be two major alterations. In the region of the seizure focus there is a marked impairment in the glutamate–glutamine cycle and elevation of intracellular glutamate content. These alterations, the molecular basis of both of which may be reduced glutamine synthetase activity, lead to chronically elevated glutamate in the extracellular ﬂuid (ECF). Glial dysfunction, as evidenced by low levels of glutamine synthetase, contributes to slow clearance of extracellular glutamate. Elevated extracellular levels of glutamate would enhance neuronal excitability and glial activation. It may contribute to the epileptogenicity of the seizure focus. Chronically increased extracellular glutamate would contribute to ongoing neuronal loss (excitotoxicity) and reactive glial proliferation. Away from the focus there is a decrease in GABA concentrations, which appears to be due to a reduction in the GABA synthesis rate. One plausible hypothesis is that below normal intracellular GABA content contributes to enhanced cortical excitability and therefore to the potential for seizures. Low cellular GABA levels reﬂect an intrinsic alteration in GABA metabolism and may not be sufﬁcient to serve as the sole cause of epilepsy in most patients. We hypothesize that low neocortical GABA levels contribute to the expression of seizure disorders in the localization-related epilepsies by increasing cortical excitability in a global fashion, thereby facilitating the spread of seizure to regions remote from the epileptogenic zone. The combination of below normal cellular GABA content and above normal cellular glutamate content may act synergistically to cause epilepsy in those patients without anatomical lesions, ‘MRI-negative’. Spectroscopic imaging of glutamate and GABA may delineate the epileptic networks responsible for seizures in these patients. Below normal GABA and homocarnosine content appears to contribute to the seizure diathesis of some patients with idiopathic generalized epilepsies, e.g., juvenile myoclonic epilepsy. In addition to the alterations of the glutamate–glutamine cycle and GABA metabolism described here there is direct evidence of widespread mitochondrial dysfunction both within and remote from the sclerotic hippocampus in MTLE (see previous chapter). Phosphocreatine to ATP (PCr/ATP) ratios are reduced to the greatest extent in the amygdala ipsilateral to the seizure focus followed by the ipsilateral pes, hippocampus, and thalamus with decreasing severity. A similar pattern was seen in the contralateral hemisphere, albeit to a lesser extent (Hetherington et al., 2002a,b). A similar pattern of loss is seen with respect to N acetyl aspartate (NAA) levels. The NAA content of brain is controlled by the rate of synthesis in neuronal mitochondria, less the rate of neuronal release or oxidation (Petroff et al., 2002a; Moreno et al., 2001). Low PCr/ATP ratios and low NAA are reported also in the mitochondrial cytopathies and are associated with cortical hyperexcitability (Kunz, 2002). Hetherington and Spencer have proposed a mechanism by which impaired neuronal mitochondrial energetics could lead to increased glutamate release, due to dysregulation

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of calcium metabolism. In combination with the impairment in the glutamate–glutamine cycle the increase in glutamate release (for a given degree of electrical activity) would explain the chronically elevated ECF glutamate and the hyperexcitability characteristic of the epileptogenic hippocampus.

Acknowledgements Grant support was provided by National Institute of Health (NINDS) PO1-NS39092. We thank our colleagues who helped design, acquired data for, and analyzed the studies including Idil Cavus, Nihal C. de Lanerolle, Tore Eid, Laura D. Errante, Hoby P. Hetherington, Fahmeed Hyder, Jung H. Kim, Richard H. Mattson, Douglas L. Rothman, and Anne Williamson. We thank the patients participating in these studies.

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13 The Role of Altered Energetics of Neurotransmitter Systems in Psychiatric Disease Graeme F. Mason Department of Psychiatry, Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

13.1 Glial–Neuronal Interactions and Neuropsychiatric Disorders 13.2 1 H MRS and Glial–Neuronal Interactions 13.2.1 N-Acetyl Aspartate 13.2.2 Glutamate, Glutamine, and GABA 13.3 13 C MRS and Psychiatric Disorders 13.4 Future Directions

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The investigation and evaluation of psychiatric disorders has for most of its history consisted largely of qualitative and subjective measurements of symptoms and behaviors. Today’s views lean strongly toward the biochemical and morphological, with investigations that use the numerous brain imaging techniques, biochemical assays of body ﬂuids and tissues, and genetic evaluations of chromosomal regions, including some speciﬁc target genes. A ﬁeld long limited to subjective evaluations of disorders and the effectiveness of treatment now can avail itself of an abundance of quantitative methods. These methods are broadly used in two ways. First, investigators hoping to understand the disorders test hypotheses about brain function, shape, blood ﬂow, genetics, or chemistry. Researchers hope that by ﬁnding alterations, such as a brain region with altered morphometry, a polymorphism of the gene for a neurotransmitter receptor, or a metabolic change, a target for rational drug design can be obtained. Second, researchers are searching for quantitative, rapidly detectible biomarkers of disease, ideally markers that are responsive to treatment. Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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Some potential biomarkers of disease and treatment efﬁcacy have been reported based on studies of genetics (Del Zompo et al., 1984; Hovatta et al., 1991; Stine et al., 1997), brain morphometry (Busatto et al., 2003; Soares, 2002), receptor binding (Dean, 2003; Varrone et al., 2001), and brain blood ﬂow and metabolism (Kumar et al., 1993b; Saxena et al., 2002). Nuclear magnetic resonance spectroscopy, commonly called MRS in the ﬁeld of psychiatric research, can make a unique contribution because of its ability to make direct measurements of brain chemicals in vivo in healthy subjects and patients before and during treatments or substance withdrawal. The ﬁndings up to the present time have revealed altered functional neurochemistry in several disorders.

13.1. GLIAL–NEURONAL INTERACTIONS AND NEUROPSYCHIATRIC DISORDERS Evidence has gradually accumulated for metabolic interactions between glia and neurons in the healthy brain. Erecinska and Silver (1990) reviewed evidence from a variety of experiments to present a scenario in which glutamate is released from glutamatergic neurons, taken up by astrocytes, and converted to glutamine. The glutamine is then returned to neurons, which convert the glutamine to glutamate for further neurotransmission. In the same review, a similar neurotransmitter cycle is presented for GABA and glutamine. Energetic support for such cycles could come from glial glycolysis (Magistretti and Pellerin, 1997; Sibson et al., 1998). 13 C MRS has been particularly useful for the evaluation of the glutamate–glutamine and GABA–glutamine neurotransmitter cycles, as well as for the coupling of cycling with energy metabolism (Patel et al., 2001; Sibson et al., 1997; Sibson et al., 1998; and Chapter 5). An interesting feature of the cycles is that the Michaelis–Menten half-saturation constants Km for several steps in the cycle may lie in the vicinity of the concentrations of substrates for each step in the cycle (Erecinska and Silver, 1990), which opens the possibility that small changes in concentration of any of the substrates could affect the overall rate of the cycle. Glial uptake of glutamate appears to operate at high afﬁnity (see reviews of Danbolt, 1994; Erecinska and Silver, 1990), but there are several other key steps in the process. After the astrocyte takes up glutamate, the conversion to glutamine is facilitated by glutamine synthetase, whose Km for glutamate is 10–15 mM (Lund, 1970), which is greater than either the cortical concentration of glutamate of 9–12 mM (Hawkins and Mans, 1983; Perry et al., 1987; Petroff et al., 1989) or the glial concentration of less than 1 mM (Lebon et al., 2002). After the glutamine is released and taken into the neuron, the conversion to glutamate occurs via phosphate-activated glutaminase, whose Km for glutamine has been reported to lie between 1 and several millimolar in the rat brain (Dennis et al., 1977; McGeer and McGeer, 1979; Weil-Malherbe, 1969), which is on the order of the brain concentration of glutamine (Hawkins and Mans, 1983). In a cycle of GABA and glutamine, the GABA-synthesizing enzyme glutamate decarboxylase lies in the range of 1 mM (Blindermann et al., 1978; Maitre et al., 1978; Wu et al., 1978), which is similar to what is believed to be the concentration of glutamate in the GABAergic neuron (Patel et al., 1974). More recently it has been shown that inhibition of glutamine transport depletes the pools of glutamate and GABA (Rae et al., 2003). Taken together, the set of metabolite concentrations and enzyme kinetics are consistent with a scenario in which alternations in glial–neuronal interactions could change the concentrations of glutamate, glutamine, and GABA, and vice versa. The comparison of substrate concentrations and Km values for neurotransmitter cycling should be placed, however, in the context of metabolic control analysis (MCA). Presumably, the substrate concentrations and cycling rates are controlled not by individual enzymatic steps but by the neuronal ﬁring requirements of the network of cells. The brain may be controlling the network of enzymatic steps to maintain a constant value for one or more crucial metabolic variables, such as the rate of the release of glutamate or the quantity of glutamate that is present in the extracellular space. Even with the MCA view, however, an alteration of enzyme function could be consistent with observable neurochemical changes during neuropsychiatric disorders.

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There are numerous studies of FDG-PET, primarily using metabolic rates that are normalized to the whole-brain metabolic rate of each subject. Kimbrell and co-workers have demonstrated that normalization can lead to completely different conclusions about the location of cerebral abnormalities (Kimbrell et al., 2002). A few studies of psychiatric disorders have reported absolute rates of glucose utilization obtained with FDG-PET, generally showing reductions in regional and global metabolic rates (Dunn et al., 2002; Kumar et al., 1993a). Some reductions have been shown to be proportionate to the severity of depression (Kimbrell et al., 2002). Normalized metabolic rates show that, relative to the rest of the brain in depression, the subgenual prefrontal cortex and the occipital cortex have lower rates of glucose utilization than are seen in healthy controls (Drevets et al., 1997, 1998). Although the occipital cortex was not evaluated further in those papers, the abnormality in the subgenual cortex was associated with a loss of volume (Botteron et al., 2002; Drevets et al., 1997). Reductions in glia have been reported in depressed subjects in the same brain ¨ region without apparent alteration of neurons (Ongur et al., 1998). Similarly, in alcohol-dependent subjects, reduced glial density and glial nuclear size (Miguel-Hidalgo et al., 2002) are associated with reductions in glucose utilization (Adams et al., 1993). Abnormalities of brain volume, metabolism and cellular density and form have been reported in a variety of neuropsychiatric conditions, and these ﬁndings are consistent with altered glial–neuronal interactions. As will be described, MRS provides a variety of tools that can be used to investigate alterations in glial–neuronal interactions in detail in vivo. The applications most relevant to investigations of glial–neuronal interactions are implementations of 1 H MRS, and many of the resulting MRS ﬁndings are consistent with alterations in the glial–neuronal interactions and changes in NAA metabolism. Theories of altered glial–neuronal interactions are consistent with the use of MRS to investigate and monitor psychiatric diseases and their treatments. In particular, 1 H MRS is particularly useful for psychiatric studies because of its high sensitivity and its safety for repeated, longitudinal studies in adults and children.

13.2. 1 H MRS AND GLIAL–NEURONAL INTERACTIONS 13.2.1. N -Acetyl Aspartate Measurements of NAA are the most common application of 1 H MRS in the brain. This application of MRS owes its widespread use to several qualities. First, the sensitivity of an MRS measurement is directly proportional to the quantity of the material present in the sample, and NAA is the most abundant neurochemical that can be detected with MRS in the brain. Furthermore, the resonance of NAA that is most often measured is that of the methyl group at 2.023 ppm, which contains three hydrogen atoms and therefore provides three times the sensitivity that would be obtained if only one hydrogen were detected. Another helpful feature of the NAA methyl resonance is that it is composed of a single peak. Many metabolites, such as glutamate and GABA, for example, have resonances that distribute the peak intensity among two or more peaks, thereby reducing the peak height and increasing the complexity of the spectral analysis. The cause of the multiplicity is J -coupling, which is a form of interaction of the detected hydrogen with other nuclei in the molecule. J -coupling not only increases complexity in the MR spectrum but can distort peak shapes and reduce peak areas for resonance with coupling. The last quality of NAA to make it useful for MRS studies of neuropsychiatric disorders is of biological interest. NAA is found primarily in glutamatergic neurons (Moffett et al., 1991; Urenjak et al., 1992) and has generally been assumed to provide a measure either of neuronal quantity or viability. Although its function is unknown, NAA is synthesized at a rate of about 1/100th the rate of glucose oxidation (Moreno et al., 2001). The fact that it is synthesized by a mitochondrial enzyme (Patel and Clark, 1979) raises the possibility that its production may be related to energy metabolism or brain function. NAA does leak slowly or is released in small quantities to the extracellular space, although the release is inconsistent with a neurotransmitter function for NAA (Taylor et al., 1994). Astroglia have transporters

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that import NAA speciﬁcally (Sager et al., 1999), and neurons take up very little (Baslow and Resnik, 1997). Aspartoacylase is expressed primarily in white matter tracts and oligodendrocytes (Bhakoo et al., 2001), which is consistent with earlier reports that NAA is associated with myelination and mitochondrial membranes (Burri et al., 1991; D’Adamo and Yatsu, 1966). These reports are consistent with a scenario in which NAA is synthesized in neurons in a process related to cellular energetics, released slowly to the extracellular environment, and degraded to aspartate and acetyl moieties in glia. In this scenario, disruptions in the neuronal–glial interactions or cellular energetics could alter the concentration of NAA. In vivo, the rate of glucose utilization and the level of NAA have been reported to be correlated (O’Neill et al., 2000). MR measurements of NAA must be evaluated with some caution because of technical complications in their interpretation. One of the most common difﬁculties arises from the use of echo times of 35 ms and less that are commonly used for MRS measurements. At these short echo delays, macromolecular resonances contribute signiﬁcantly to the peak intensity at the spectral locations of creatine, NAA, glutamate, glutamine, and GABA (Behar and Ogino, 1993; Behar et al., 1994; Hofmann et al., 2001; Hwang et al., 1996; Kassem and Bartha, 2003; Mader et al., 2002; Rothman et al., 1993), and their regular, reproducible line shapes are not properly ﬁtted with the normal spline baseline routines that are normally employed. The macromolecular resonances decay rapidly as echo times lengthen, whereas the NAA resonance is much more resistant to lengthening echo delays. Therefore, it is advisable to use longer echo times to study NAA. A further complication is that the glutamate, glutamine, and GABA resonances appear strongly at short echo delays, and they are unresolved from one other and usually from NAA at the commonly used magnetic ﬁeld strength of 1.5 T. NAA has been evaluated in a variety of psychiatric conditions, but the most common application is to schizophrenia. Investigators have generally reported lower NAA than is seen in healthy control subjects, with the reductions localized to speciﬁc brain regions, especially the prefrontal cortex (Bertolino et al., 2001; Buckley et al., 1994; Ende et al., 2000). One thoughtful approach combined MRS and 15 O PET to relate NAA measurements to function in schizophrenia (Bertolino et al., 2000). First, NAA was measured in different brain regions to identify the prefrontal cortical location of the abnormality in a group of patients, relative to a group of healthy control subjects. Then, 15 O PET was used to measure cerebral blood ﬂow at rest and during a cognitive task in the same subjects. In the patients, the region of low NAA showed a signiﬁcantly smaller increase in blood ﬂow during the functional task, compared to the healthy control subjects, thereby demonstrating the low NAA coincided with a functional abnormality. In schizophrenia, a mutation in brain-derived neurotrophic factor (BDNF) has been identiﬁed in patients and their unaffected relatives (Egan et al., 2003). BDNF is believed to be involved in the cellular processes of memory creation (Lu and Gottschalk, 2000; Poo, 2001), and so the investigators tested the working memory of carriers and non-carriers of the mutation (Egan et al., 2003). Working memory is the short-term retention of information to perform cognitive tasks. Homozygous carriers of the mutation had performance scores of working memory that approached the values of schizophrenic patients. The same subjects were studied with MRS to measure NAA levels, and not only did the patients and siblings of patients have signiﬁcantly reduced NAA levels, but there was a signiﬁcant effect of the BDNF mutation on the NAA levels (Egan et al., 2003). NAA has not been studied as widely in mood disorders, perhaps because the disorders are less clearly diagnosed than schizophrenia or alcohol dependence. Indications are that NAA levels are not signiﬁcantly altered in major depressive disorder (Farchione et al., 2002; Kusumakar et al., 2001). NAA may be reduced in bipolar disorder (Castillo et al., 2000; Cecil et al., 2002; Winsberg et al., 2000), which is interesting when taken in the context of similarities to schizophrenia regarding some similarity in cognitive deﬁcits. Lithium, which has contributed effectively to the treatment of bipolar disorder, has been reported to increase NAA concentrations (Moore et al., 2000).

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Some reductions of NAA levels have also been reported in alcohol dependence (Bendszus et al., 2001; Jagannathan et al., 1996; Parks et al., 2002; Schweinsburg et al., 2001). As has been discussed in this chapter, NAA appears to have a relation to neuronal function, but it may also be related to neuronal volume. In alcohol dependence, genetic factors, acute ethanol toxicity, and chronic tissue damage characterized by cortical shrinkage may all contribute to loss or shrinkage of neurons and, therefore, to reductions in NAA. The inconsistencies of the ﬁndings may result from differences in the ages, severity and duration of drinking of the subjects, nutritional state, or the presence of comorbid psychiatric disorders and other substance abuse. Perhaps more important than the discrepancies of reduction is that in at least some cases, the NAA level can increase following detoxiﬁcation (Martin et al., 1995; Parks et al., 2002), offering hope for some degree of neuronal and functional recovery and showing a link between NAA increases and cognitive improvements that are generally observed with recovery from alcohol dependence. The effects of other substances of abuse have not been as widely studied as those of ethanol, although results to date generally show reductions in NAA. Methamphetamine (Nordahl et al., 2002) and heroin (Haselhorst et al., 2002) are both associated with reductions of NAA levels, and reductions observed with ecstasy (Reneman et al., 2002) are correlated with scores on working memory testing (Reneman et al., 2001). In summary, NAA is reduced in many psychiatric disorders, and it appears that NAA may be affected by either neuronal volume or function.

13.2.2. Glutamate, Glutamine, and GABA Glutamate and GABA are neurotransmitters that are known to participate in functionally related cycles with glutamine, so these three neurochemicals are more directly linked to neuronal function than any other compound that is detected with MRS. They are also among the most difﬁcult chemicals to measure with 1 H MRS in vivo. The difﬁculties arise partly because J -coupling complicates their resonances, so that instead of well-resolved single peaks the signals are distributed among triplet resonances or in some cases even more complex patterns. Figure 13.1 shows examples of the complicated patterns for glutamate and glutamine. Furthermore, many of the resonances of glutamate, glutamine, and GABA in vivo overlap with one another or with other peaks. For that reason, most studies report peak areas for a single, irregular combined resonance in the vicinity of 2.4 ppm called ‘Glx’. At the short echo delays normally used to detect Glx, macromolecules make a major contribution to the peak intensity at glutamate, glutamine, and GABA locations (Behar and Ogino, 1993; Behar et al., 1994; Hofmann et al., 2001; Hwang et al., 1996; Kassem and Bartha, 2003; Mader et al., 2002; Rothman et al., 1993). Although the macromolecular resonances decay at long echo times, they still contribute signiﬁcantly to glutamate and glutamine at the echo delay of 30 ms that is generally employed for psychiatric 1 H MRS studies. Because of the issues of resolution, signal-to-noise ratios, and macromolecular contamination, many of the glutamate and glutamine measurements made in psychiatric research should be viewed with skepticism. Before discussing psychiatric applications of MRS measurements of glutamate and GABA, it may be helpful to provide some context with regard to functional effects on neurotransmitter levels. It is thought that cortical excitability is a reﬂection of cortical GABAergic tone, so by modulating the GABAergic tone, one would expect to affect the ability to excite cortical neurons. Indeed, transcranial magnetic stimulation has been used to measure the voltage threshold for somatosensory excitation in the human brain before and after peripheral electrical stimulation in a nerve at the wrist. After stimulation, the voltage threshold for cortical excitation was increased (Kaelin-Lang et al., 2002). The effect was blocked by lorazepam, which is a GABAA receptor agonist. With light deprivation, a similar increase in cortical excitability in the occipital lobe was observed during 45 min of darkness (Boroojerdi et al., 2000a), and the effect was accompanied by a 20 % drop in GABA levels over the same period (Boroojerdi et al., 2000b). No information about

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Glutamine 270 ms

130 ms

70 ms

50 ms

20 ms

15 ms

10 ms 4.00

3.50

3.00 2.50 2.00 PPM

4.00

3.50

3.00 2.50 2.00 PPM

Figure 13.1. The effect of J -modulation on the resonance intensity and line shape of glutamate and glutamine at a magnetic ﬁeld strength of 4.1 T. The data were acquired from a solution of 50 mM glutamate and 50 mM glutamine dissolved in distilled water, using a spin-echo sequence with a range of echo delays. As the echo time increased, the J -modulation reduced the resonance intensities rapidly, and although the modulation caused some return to coherence at TE = 130 and 270 ms, the modulated return was incomplete. (Mason et al., 1996 Metabolic Brain Disease. vol. 11, p. 283–313.)

the concentration of glutamate was obtained in those studies, but these studies suggest that in conditions of reduced GABA levels, one might speculate that glutamate release is reduced. The glutamatergic and GABAergic systems have been implicated in the pathophysiology of schizophrenia from diverse lines of evidence. NMDA receptor function may be altered in schizophrenia (Javitt and Zukin, 1991; Olney and Farber, 1995). Pharmacological studies have shown that NMDA receptor antagonists can exacerbate symptoms of schizophrenia (Javitt and Zukin, 1991; Krystal et al., 1994; Malhotra et al., 1996), while reducing glutamate release with lamotrigine attenuates psychotic effects associated with NMDA receptor antagonism (Anand et al., 2000). Examining the glutamate–glutamine and GABA–glutamine cycles from an enzymatic perspective, the prefrontal cortex of patients with schizophrenia have been shown to possess four times the activity of phosphate-activated glutaminase and twice the activity of glutamate decarboxylase (GAD-65 and GAD-67 combined) (Gluck et al., 2002). Other studies showed decreased messenger RNA for GAD-67 in the prefrontal cortex of schizophrenic patients (Abkarian et al., 1995; Volk et al., 2000), and the loss of some cortical GABAergic neurons (Reynolds et al., 2002). One possible reconciliation of the reports is that most of the increased GAD in schizophrenia is GAD-65, instead of GAD-67. GAD-67 is located primarily in the cell bodies, while GAD-65 is found in the nerve terminals, so such an explanation would suggest that in schizophrenia GABA synthesis is up-regulated in the nerve terminals. There is also a report, however, of reduced GABA release and uptake in schizophrenia (Simpson et al., 1989) and a further observation of upregulated GAD-65 in association with antipsychotic treatment (Benes et al., 2000). The ﬁndings of GABAergic and glutamatergic alterations have typically been

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Cr/PCr

Glx C3 Glu C4 NAA Gln C4

245

NAA

Cho

Difference: just metabolites

GABA (buried)

Total signal Macromolecules 4.0

3.5

3.0 ppm

2.5

2.0

1.5

Figure 13.2. The overlap of GABA with glutamate, glutamine, and other resonances in spectra obtained at 2.1 T. The middle spectrum shows the spectrum obtained with the conventional spin-echo sequence using an echo delay of 12 ms. If an inversion–recovery sequence is applied to null the metabolite resonances, only the macromolecular resonances remain (bottom). When the macromolecular contribution is subtracted from the total signal, only the metabolites remain (top). The macromolecular signal can contribute signiﬁcantly to the total signal amplitude and needs to be considered when quantifying glutamate and glutamine in vivo.

hypothesized as defects in glutamatergic and GABAergic systems (Gluck et al., 2002), but it is possible that the primary defects in schizophrenia lie elsewhere, and the changes in the glutamatergic and GABAergic systems are either adaptive or maladaptive strategies by the brain to compensate for the primary defect. No matter where the defects lie, it appears likely that changes may occur in the glutamatergic and GABAergic pathways, and MRS is one tool to investigate those changes. The technical difﬁculties in the measurement of glutamate, glutamine, and GABA described earlier in this section have prevented most groups from investigating these important metabolites in schizophrenia. A study at 1.5 T reported increased Glx in schizophrenic patients and speculated that Glx might be elevated due to increased glutamine as a result of above normal glutamate release (Bartha et al., 1997). In a more recent study, the same group at a magnetic ﬁeld strength of 4 T reported that the increase is, in fact, glutamine, without a concomitant change in glutamate (Th´eberge et al., 2002). Little else has been reported for glutamate, glutamine, and GABA in schizophrenia with MRS. At 4 T, the spectral resolution is adequate to differentiate the glutamate and glutamine, but a cautionary note is that at the 20 ms echo delay used in this study, macromolecules are a signiﬁcant contaminant of the glutamate and glutamine peaks (Figure 13.2). GABA has been implicated strongly in mood disorders (Chang et al., 2003; Sanacora et al., 2000), with reported alterations in GABA receptors (Cheetham et al., 1988; Gulinello et al., 2003; Kaschka et al., 1995), plasma GABA levels (Petty et al., 1992), brain GABA levels (Baker et al., 1991; Sanacora et al., 1999), and beneﬁcial effects of GABA receptor agonists (Borsini et al., 1986, 1988; Delini-Stula, 1987). A study of anxiety disorder yielded a report of elevations of Glx orbitofrontal cortex, using an echo delay of 12 ms (Grachev and Apkarian, 2000). Although the spectra presented are of good quality, no macromolecular corrections were made, so some caution must be applied in the interpretation of the results. Another study using J -editing (Figure 13.3) to detect GABA showed that cortical GABA levels are lower in patients with panic disorder (Goddard et al., 2001). This combination of studies, one of Glx, the other of GABA, suggests the possibility that the brain maintains GABA and glutamate levels in a reciprocal relationship. That is, when one increases, the other decreases. A reciprocal relationship is also suggested by recent measurements in major depressive disorder, where the concentration of GABA has been shown to be reduced by 30–50 % (Sanacora et al., 1999) and the level

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Brain Energetics and Neuronal Activity NAA

Anxiety diff (x4) GABA

Control diff (x4) Control with DANTE

Cho 5

4

Cr

3 2 Chemical shift scale, ppm

Control without DANTE 1

0

Figure 13.3. Representative γ -aminobutyric acid (GABA) spectra from a patient with panic disorder (top spectrum) and a control subject (second from top). The patient has signiﬁcantly less GABA than the control subject. The bottom two spectra provide a demonstration of the J -editing of GABA in vivo. For J -editing, two spectra are acquired for each measurement of GABA. One spectrum has GABA, creatine, and choline in phase with one another, and the other spectrum has GABA inverted with respect to the creatine and choline, using a DANTE pulse. Upon subtraction, the creatine and choline resonances vanish, because they are identical, whereas the GABA adds to itself because its phase differs between the two spectra. (Goddard et al., 2001).

of glutamate increased by 10 % (Mason et al., 2001a). In the context of reports of reduced glucose utilization in major depressive disorder (Dunn et al., 2002; Kimbrell et al., 2002; Kumar et al., 1993b), it may be that the low GABA is related to chronically decreased glutamate release, just as was the case with the light deprivation (Boroojerdi et al., 2000b). The reductions in GABA were normalized with electroconvulsive therapy (ECT) (Sanacora et al., 2003) or treatment with selective serotonin reuptake inhibitors (Sanacora et al., 2002), although the number of subjects was too small to evaluate the effects of the treatments on the level of glutamate. The increase of GABA concentration with ECT is accompanied by an increase in GABA receptor binding after ECT (Mervaala et al., 2001), implying an alteration in multiple parts of the GABAergic system. In contrast to major depressive disorder, manic-depressive disorder does not show alterations in either GABA or glutamate. Although the glutamatergic system has also shown alterations in mood disorders (Krystal et al., 2002), little has been reported about concentrations of glutamate, glutamine, and GABA with MRS in vivo in mood disorders. Another area of investigation of 1 H MRS studies of mood disorders lies with neurosteroid modulation of the GABAergic and glutamatergic systems. Neurosteroids are known to bind to the GABA receptor and potentiate the GABAergic response (Puia et al., 2003; Smith et al., 1998), and plasma GABA levels have been reported to vary across the menstrual cycle (Halbreich et al., 1996) and to be lower in women with premenstrual dysphoric disorder (PMDD). Cortical GABA levels have also been shown to vary across the menstrual cycle but in a manner that differs in women with PMDD, relative to healthy control subjects (Epperson et al., 2002). That is, in the follicular phase, during which healthy women have the highest levels of brain GABA, women with PMDD have the lowest GABA concentrations. Furthermore, glutamate levels are elevated when GABA levels are decreased (Haga et al., 2001), which again raises the possibility of a reciprocal relationship of GABA and glutamate concentrations. The GABAergic system is immediately suspected of involvement in alcoholism because of the wellknown potentiation of the GABAA receptor by the binding of ethanol. Further evidence for GABAergic involvement comes from low plasma GABA levels in alcohol-dependent subjects (Petty et al., 1993) and some efﬁcacy of the GABA receptor agonist baclofen in reducing alcohol consumption (Addolorato et al.,

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2000, 2002; Colombo et al., 2000). GABA levels in alcohol-dependent subjects after 1 month of sobriety have been shown to be reduced by ∼25 % compared to healthy control subjects (Behar et al., 1999). More recently, GABA levels measured by 1 H MRS have been found to be normal after 1 week of detoxiﬁcation but fall by approximately 20 % after 4 weeks of sobriety (Mason et al., 2003a), which is in agreement with a signiﬁcant decline in plasma GABA levels between 1 day and 3 weeks of sobriety (Adinoff et al., 1995).

13.3. 13

13

C MRS AND PSYCHIATRIC DISORDERS

C MRS studies have focused primarily on the metabolism of glutamate, glutamine, and GABA, with some attention devoted to NAA. Unlike NAA, the amino acids glutamate, glutamine and GABA are synthesized relatively rapidly in the brain, with the entire quantities turned over in about 1 h. In vivo, the use of glucose labeled at the C1 position has yielded the rates of glucose oxidation, glutamate–glutamine neurotransmitter cycling, GABA synthesis, neuronal α-ketoglutarate–glutamate exchange, and the neuronal TCA cycle (Gruetter et al., 2001; Mason et al., 1992, 1995, 2001b; Sibson et al., 1997, 1998). Glucose labeled at C2 has provided rates of anaplerosis and estimates of the astrocytic TCA cycle ﬂow relative to the rate of glutamine synthesis (Kanamatsu and Tsukada, 1999; Lebon et al., 2002; Tsukada et al., 1998). Acetate labeled at C1 has yielded the rate of the astrocytic TCA cycle (Bl¨uml et al., 2002), while acetate labeled at C2 has provided estimates of glutamate–glutamine neurotransmitter cycling and the astrocytic glutamate pool size and astrocytic TCA cycle rate (Lebon et al., 2002). In cellular preparations, even compartmentation of intermediary metabolism within glia can be evaluated (Zwingmann et al., 2001). At high magnetic ﬁeld strengths, the greater sensitivity allows the deconvolution of partial volume effects on metabolic rates (Mason et al., 1999). The richness of the information available with 13 C permits the study of basic mechanisms of glial–neuronal interactions and, therefore, neuropsychiatric diseases in which these may be affected. Because 13 C MRS is relatively new for human use, it is only now beginning to be applied to psychiatric disorders, with the ﬁrst preliminary results obtained in an investigation of the low concentration of cortical GABA (Mason et al., 2001c). As described earlier in this chapter, GABA has been reported to be reduced by 30–50 % in depressed subjects (Sanacora et al., 1999). In light of reductions observed in glucose utilization in major depression (Dunn et al., 2002; Kimbrell et al., 2002; Kumar et al., 1993a), at least two theories have been proposed to explain the GABA reduction: (1) the GABAergic system is more active than normal, releasing an abundance of GABA, inhibiting neuronal ﬁring, and depleting the level of GABA to a new lower steady state, and (2) the GABAergic system has been slowed because neuronal activity is globally lower. In the ﬁrst case, the rate of GABA synthesis should be elevated in depressed subjects, but in the second case, the rate of GABA synthesis should be slower (Figure 13.4). The early results obtained using infusions of [1-13 C]glucose in depressed patients indicate that the rate of GABA synthesis is approximately 50 % of the rate seen in healthy controls (Mason et al., 2001c), which is consistent with the reported reductions in GABA concentration. The basic ﬁnding is that GABA synthesis and, presumably release, are slower in depression, and the 13 C MRS has advanced the ﬁeld to the question of why GABA and possibly glutamate release are reduced in depression. 13 C can be used productively in vivo without regard to kinetics of neurotransmitter metabolism. At least three alternative applications have been achieved. The ﬁrst of these is simply a measurement of the concentrations of glutamate and glutamine to overcome the technical difﬁculties associated with 1 H MRS detection of glutamate and glutamine. The natural abundance 13 C resonances of glutamate and glutamine C4 at 1.5 T provide more than adequate resolution to quantify glutamate and glutamine. The approach was used to compare healthy children to an infant with Canavan’s disease, demonstrating reduced glutamate and increased NAA in the child with Canavan’s disease (Bl¨uml, 1999). A second approach overcomes some of the low sensitivity of 13 C MRS while maintaining the high spectral resolution, and

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Brain Energetics and Neuronal Activity Gln2 Glu2

NAA2 Asp2

60

50

Glu3

Glu4 Asp3

Gln4

Gln4 Gln2

Gln3

Glu2

Glu3 Gln3

Asp2

NAA3

40

Asp3 Glu4

30

20

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Figure 13.4. 13 C label incorporation in control (left) and a representative patient with hepatic encephalopathy, grade I–II (right). The spectra were calculated from one acquisition, 80–100 min after start of an infusion of [1-13 C]glucose infusion. The spectra were scaled to represent absolute concentrations. Note the apparent reduction of 13 C incorporation into glutamate C4 and C2, and aspartate C2 and C3 in CHE. Even though affected by the subtraction analysis to a larger extent in CHE than in control, the glutamine C4 resonance appears strikingly prominent in spectra of the patient. Label accumulation in NAA C2 and C3 observed in the control was not detected in CHE. (Bl¨uml et al., 2001). Reproduced with permission of John Wiley & Sons, Inc.

Glutamatergic neuron

Glia

Glutamate 3 Glutamine

Glutamate

Glutamate 2 GABA 1 GABAergic neuron

Figure 13.5. Cycling of glutamate, GABA, and glutamine. In depression, the concentration of cortical GABA is low. The reduction could be explained on the basis of GABA release (labeled 1) being elevated in depression and draining the GABA pool to a new steady-state level that can be sustained by glutamate conversion to GABA (labeled 2). Another possibility is that glutamate release (labeled 3) is slowed down in depression, so that less GABA is released (labeled 1), and a new higher steady-state concentration is reached. Preliminary data obtained with 13 C indicate that the rate of GABA synthesis is reduced, which is consistent with the second scenario.

that is achieved by measuring concentrations of 13 C-labeled glutamate and glutamine after an infusion of [1-13 C]glucose. This approach has been used successfully to relate labeled quantities of glutamine with disease grade in hepatic encephalopathy (Bl¨uml et al., 2001) (Figure 13.5). Of note is that NAA labeling in the patients was much lower than in the healthy controls, demonstrating that reduced NAA turnover rates can be detected in some conditions of neuronal compromise. These are two practical applications that are particularly useful for the evaluation of glutamate and glutamine at the low magnetic ﬁeld strengths that are in widespread use. A third application is the measurement of the rate of synthesis of NAA (Moreno et al.,

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2001) during an infusion of [1-13 C]glucose. In light of the possibility of glial–neuronal mediation of NAA turnover, measurements of the rate of NAA turnover may prove useful for studying psychiatric disorders that are associated with reduced levels of NAA and in which glial-neuronal interactions are hypothesized to be abnormal.

13.4. FUTURE DIRECTIONS 1

H MRS studies yield measurements of the concentrations in altered disease or functional states. Those static chemical measurements are material for the development of hypotheses about neuronal viability, glial–neuronal interactions, and brain energetics. Because glutamate and GABA are the most abundant neurotransmitters in the brain, the widespread ability to make robust and accurate measurements of concentrations of glutamate, glutamine, and GABA would be of enormous beneﬁt to research on psychiatric disorders. The rapidly spreading use of high-ﬁeld magnets for MR spectroscopy will provide users with enhanced opportunities to overcome the present, serious limitations of spectral resolution, sensitivity, and macromolecular contamination. The number of groups that are eliminating the macromolecular contamination in 1 H MRS is increasing, and it should soon become standard practice. Although 1 H MRS has provided abundant information about neuropsychiatric disorders, more work is required for the evaluation of neurotransmitter hypotheses that result from the MRS studies, and 13 C MRS will play a major role. Among current hypotheses for alterations in 1 H MRS concentrations of brain metabolites are alterations of energy metabolism, glutamate and GABA neurotransmitter release, glial–neuronal interactions, and the number and volume of neurons. One example of the evaluation of a hypothesis resulting from 1 H MRS measurements of the lower concentration of GABA in depression is the 13 C ﬁnding of slow GABA synthesis. 13 C MRS studies have powerful beneﬁts, such as a lack of radioactivity, high spectral resolution, and the ability to measure the rates of several metabolic pathways at the same time. The lack of radioactivity in particular opens the possibility of studying children, whose disorders have generally been neglected in psychiatric research. However, the disadvantages may preclude some disorders from study on the basis of practicality. The infusion times are long, typically 2 h. If absolute measurements of glucose utilization are needed, 15 min experiment times have recently been developed in animals using [2-13 C]deoxyglucose (Cohen et al., 2002). For other rates, such as GABA synthesis or glutamate–glutamine cycling, longer infusion times are currently required. Higher-ﬁeld magnets and the accompanying improvements in sensitivity will help to decrease the required experiment time and thereby expand the types of disorders and the numbers of patients who are willing and able to take part in 13 C research projects. A practical obstacle to patient compliance is the need for two intravenous (IV) catheters, one of which is used to infuse the 13 C-labeled substrate and the other to draw blood samples. The greatest sensitivity is obtained when the 13 C enrichment of the substrate in the plasma changes as rapidly as possible, and the most rapid changes available are obtained with IV infusions. One study has used a slower enrichment method, which was oral administration of [1-13 C]glucose to measure the rate of synthesis of NAA (Moreno et al., 2001). Because the rate of synthesis of NAA is so very much slower than even the slow rate of glucose enrichment from the gut, the measurement retained practically full sensitivity. One IV catheter was eliminated by the oral administration. For measurements of more rapid processes such as glucose oxidation and glutamate–glutamine neurotransmitter cycling, however, a comparison of oral and IV administration at 2.1 T showed a major loss of sensitivity for such rapid processes when oral administration is used (Mason et al., 2003b). Perhaps adequate sensitivity can be recovered by doing measurements at higher magnetic ﬁeld strengths. The study of NAA synthesis also eliminated the second IV catheter by creatively using observed glutamate and glutamine labeling as input functions for the NAA synthesis determination (Moreno et al., 2001), which is not feasible for studies of glutamate and glutamine neurotransmitter cycling and GABA synthesis.

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Under some conditions, it may be possible to eliminate the sampling IV line, as well. For IV glucose infusions, it will continue to be necessary to maintain a sampling IV catheter to ensure that plasma glucose levels remain within safe bounds. However, it may become possible to measure the time courses of 13 Clabeled glucose and the total glucose levels in the brain during oral administration of glucose, so that the percent enrichment could be observed simultaneously with the labeling of brain glutamate and glutamine. Then, the sampling IV line could be eliminated, which would greatly improve the appeal of the procedure to patients and control subjects alike. In summary, 1 H MRS is beginning to yield data that suggest alterations in glial–neuronal interactions through NAA and neurotransmitter metabolism. A new family of methods, 13 C MRS, is being applied to evaluate those interactions more directly. The increasing ﬁeld strengths and dissemination of methodological improvements should create an environment where widespread, good quality investigation of glial-neuronal interactions can provide biomarkers so eagerly sought by industry and yield abundant, valuable information to understand psychiatric disorders.

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Schweinsburg, B. C., Taylor, M. J., Alhassoon, O. M., Videen, J. S., Brown, G. G., Patterson, T. L., Berger, F., and Grant, I. (2001) Chemical pathology in brain white matter of recently detoxiﬁed alcoholics: a 1H magnetic resonance spectroscopy investigation of alcohol-associated frontal lobe injury. Alcohol Clin Exp Res 25: 924–934 Sibson, N. R., Dhankhar, A., Mason, G. F., KL, K. L. B., Rothman, D. L., and Shulman, R. G. (1997) In vivo 13C NMR measurements of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling. Proc Natl Acad Sci USA 94: 2699–2704 Sibson, N. R., Dhankhar, A., Mason, G. F., Rothman, D. L., Behar, K. L., and Shulman, R. G. (1998) Stoichiometric coupling of brain glucose metabolism and glutamate cycling. Proc Natl Acad Sci USA 95: 316–321 Simpson, M. D., Slater, P., Deakin, J. F., Royston, M. C., and Skan, W. J. (1989) Reduced GABA uptake sites in the temporal lobe in schizophrenia. Neurosci Lett 107: 211–215 Smith, S. S., Gong, Q. H., Hsu, F., Markowitz, R. S., French-Mullen, J. M., and Li, X. (1998) GABAA receptor α-4 subunit supperssion prevents withdrawal properties of an endogenous steriod. Nature 392: 3239–3244 Soares, J. C. (2002) Can brain-imaging studies provide a ‘mood stabilizer signature?’ Mol Psychiatr 7: S64–70 Stine, O. C., McMahon, F. J., Chen, L., Xu, J., Meyers, D. A., MacKinnon, D. F., Simpson, S., McInnis, M. G., Rice, J. P., Goate, A., Reich, T., Edenberg, H. J., Foroud, T., Nurnberger, J. I. J., Detera-Wadleigh, S. D., Goldin, L. R., Guroff, J., Gershon, E. S., Blehar, M. C., and DePaulo, J. R. (1997) Initial genome screen for bipolar disorder in the NIMH genetics initiative pedigrees: chromosomes 2, 11, 13, 14, and X. Am J Med Genet 74: 263–269 Taylor, D. L., Davies, E. C., Obrenovitch, T. P., and Urenjak, J. (1994) Extracellular N-acetylaspartate in the rat brain: in vivo determination of basal levels and changes evoked by high K+ . J Neurochem 62: 2349–2355 Th´eberge, J., Bartha, R., Drost, D. J., Menon, R. S., Malla, A., Takhar, J., Neufeld, R. W., Rogers, J., Pavlosky, W., Schaefer, B., Densmore, M., Al-Semaan, Y., and Williamson, P. C. (2002) Glutamate and glutamine measured with 4.0 T proton MRS in never-treated patients with schizophrenia and healthy volunteers. Am J Psychiatr 159: 1944–1946 Tsukada, Y., Kanamatsu, T., Watanabe, H., and Okamoto, K. (1998) In vivo investigation of glutamate-glutamine metabolism in hyperammonemic monkey brain using 13C-magnetic resonance spectroscopy. Dev Neurosci 20: 427–433 Urenjak, J., Williams, S. R., Gadian, D. G., and Noble, M. (1992) Speciﬁc expression of N-acetylaspartate in neurons, oligodendrocytes in vitro. J Neurochem 59: 55–61 Varrone, A., Marek, K. L., Jennings, D., Innis, R. B., and Seibyl, J. P. (2001) [(123)I]beta-CIT SPECT imaging demonstrates reduced density of striatal dopamine transporters in Parkinson’s disease and multiple system atrophy. Movement Disorders 16: 1023–1032 Volk, D., Austin, M. C., Pierri, J. N., Sampson, A. R., and Lewis, D. A. (2000) Decreased glutamatic acid decarboxylase67 messenger RNA expression in a subset of prefrontal cortical γ -aminobutyric acid neurons in subjects with schizophrenia. Arch Gen Psychiatr 57: 237–245 Weil-Malherbe, H. (1969) Activators and inhibitors of brain glutaminase. J Neurochem 19: 2257–2267 Winsberg, M. E., Sachs, N., Tate, D. L., Adalsteinsson, E., Spielman, D., and Ketter, T. A. (2000) Decreased dorsolateral prefrontal N-acetyl aspartate in bipolar disorder. Biol Psychiatr. 47: 475–481 Wu, J.-Y., Chude, O., Wein, J., Roberts, E., Saito, K., and Wong, E. (1978) Distribution and tissue speciﬁcity of glutamate decarboxylase (EC 4.1.1.15). J Neurochem 30: 849–857 Zwingmann, C., Richter-Landsberg, C., and Leibfritz, D. (2001) 13C isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes. Glia 34: 200–212

Section D Brain and Mind

14 Long-term Memory: Do Incremental Signals Reﬂect Engagement of Cognitive Processes? Jed A. Meltzer and R. Todd Constable Department of Diagnostic Radiology, Yale University School of Medicine, MR Center, P.O. Box 208042, New Haven, CT 06520-8042, USA

14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 14.10

Introduction Interpretive Methodology in Neuroimaging Long-term Memory: Broad Anatomical Dissociations MTL Involvement in Declarative Memory Encoding Factors Related to Encoding Reality of Encoding as a Cognitive Process Encoding Versus Retrieval Subdivisions of MTL and Long-term Memory: Correspondences Imaging Studies of Episodic Recall Versus Familiarity Summary

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14.1. INTRODUCTION Long-term memory is a general property of mammalian nervous systems that deﬁes simple classiﬁcation. Like other primary domains of cognitive science, such as attention and language, LTM has been the topic of dozens of neuroimaging studies designed to elucidate its basic neural mechanisms and their anatomical localization. Although the ability of fMRI to map physiological changes associated with cognitive activity in the awake human has proven enormously useful, controversy over the interpretation of such changes has Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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persisted throughout the lifetime of the ﬁeld. Interpretation of neuroimaging data involves uncertainty on many levels, from the biophysical source of the signal, to the nature of the theoretical mental processes that one hopes to isolate through cognitive task design. In this chapter, we review the progress that has been made through neuroimaging studies towards an understanding of the neural basis of long-term memory. We focus on ﬁndings of activation in the medial temporal lobe (MTL), which has long been known to be necessary for certain kinds of long-term memory function. Although neural activity in the MTL undoubtedly plays a vital role in the establishment of long-term memories and their subsequent recall, neuroimaging studies have generally failed to detect any incremental signal changes in the MTL related to the performance of tasks designed to invoke mnemonic processing, in comparison to a ‘baseline’ state. However, signiﬁcant changes have been observed in experiments that compare activity under different conditions, which, while identical in terms of the physical nature of stimulus presentation and response demands, differ with respect to incidental factors such as stimulus novelty, meaningfulness, and the extent to which the stimuli are subsequently remembered after scanning. While these various factors may reﬂect a common underlying process related to memory formation, the relationship between the hypothesized processes of memory encoding and retrieval on the one hand, and observable incremental signal changes on the other, has proven to be more indirect and nuanced than expected. This pattern of ﬁndings illustrates the difﬁculties involved in attempts to establish direct links between the neuroenergetic changes observable in functional neuroimaging and the engagement of mental processes whose physical instantiation is largely uncertain. In reviewing neuroimaging studies that have detected activation in the MTL, we consider the extent to which the factors inﬂuencing signal levels there can be construed as reﬂecting differential engagement of the theoretical cognitive processes of memory encoding and retrieval. While the data are consistent with the participation of the MTL in these processes, they suggest that incremental changes in neuronal energy use, along with the associated hemodynamic changes detectable in fMRI, are not easily evoked in the MTL by the deliberate performance of a cognitive task. This does not imply that the MTL is only involved in processing the distinctions that do drive signal changes, but rather that its normal functions in memory may not require incremental increases in neural activity across different behavioral states. The subtle and intriguing factors to which MTL activation has proved sensitive might never have been discovered, if comparisons with simple baseline tasks had turned out to be effective at producing signiﬁcant signal changes. Therefore, the results of long-term memory research may prove instructive for those seeking to interpret signal changes, or lack thereof, in other brain regions, as they demonstrate no straightforward relationship between a cognitive process and the presence of an incremental signal change.

14.2. INTERPRETIVE METHODOLOGY IN NEUROIMAGING If the component processes that make up attention, memory, and language are to be successfully localized and distinguished using fMRI, the validity of the assumptions used must be carefully considered. The ﬁrst, and most valuable, is that incremental changes in the signal (typically BOLD) reﬂect changes in local neural activity. Biophysical testing of this hypothesis, described fully in Chapters 9 and 16 of this volume, indicates that the magnitude of BOLD signal (expressed as percent change) can provide measures of the percent change in the cerebral metabolic rate of oxygen consumption (CMRO2 ), provided that measurements are available of all the other parameters that determine the BOLD signal, including cerebral blood ﬂow, cerebral blood volume, and NMR spin relaxation parameters. As described in Chapters 5 and 10, the energy increments (CMRO2 ) are directly proportional to percent changes in neuronal ﬁring rates and glutamate release. Hence, a BOLD signal change can be inferred to reﬂect a change in the level of neuronal activity. However, experiments calibrated in terms of CMRO2 (Shulman et al., 2002) have indicated that the increments of localized neuronal activity induced by sensory stimulation in awake

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animals are small relative to a large baseline level of energy use, the function of which is poorly understood. Furthermore, the neural processing of sensory stimulation of a given magnitude requires a consistent total amount of neuronal activity, rather than a consistent increment from a variable baseline (Hyder et al., 2002). Given these ﬁndings, the comparative magnitudes of BOLD signal changes obtained under different conditions can be interpreted only in very limited circumstances. When only the magnitude of a sensory stimulus is varied, the size of the BOLD signal change within a brain area can monotonically reﬂect the magnitude of the difference in neuronal activity. However, comparisons of the magnitudes of signal changes across different tasks and locations cannot be considered to be meaningful, as the size of the increment is entirely dependent on the precise nature of the baseline with which it is compared. The incremental signals observed in subtractive analyses provide evidence that a given brain area is engaged in information processing induced by the task, but failure to detect such a change is by no means proof that the neural activity occurring in an area does not play a role in the task, as the baseline amount of activity may be sufﬁcient for the region’s contribution. Consider an analogous case from the ERP literature. The ERPs to brief auditory stimuli, such as beeps, include a series of early components known to be generated by brainstem auditory structures (Rugg and Coles, 1995). The presentation of a ‘rare’ beep, differing slightly in frequency from the majority, gives rise to a ‘P300’ response – a more positive voltage level in the time-locked average response to the rare tones compared to the majority tones, occurring approximately 300 ms after the beep. This response is known to be cortical in origin (Soltani and Knight, 2000). Nonetheless, it would be absurd to conclude that the brainstem auditory components are ‘not involved’ in processing the difference between the rare and majority tones. In many ERP studies, including those of the P300 effect, the ﬁnding of interest is not that a component is present in one condition and absent in another, but rather that a deﬂection from the prestimulus baseline occurring at a certain time is larger in one condition. Therefore, it is necessary to use conditions that are exactly matched in terms of the physical nature of the stimuli and the response, so that the appropriate ‘components,’ i.e. deﬂections with a known time course, can be reliably elicited in both conditions and their magnitude compared. In fMRI studies of long-term memory, the same technique has proven absolutely essential to elucidating the role of the medial temporal lobe, as comparisons between different task states have seldom yielded positive results. However, the use of constant task demands of visual stimulus presentation has revealed that the magnitude of activity evoked in MTL structures (such as that measured relative to an average prestimulus baseline in an event-related paradigm) is sensitive to certain cognitive factors, even though two conditions may both evoke a signiﬁcant response. Therefore, the magnitude of an fMRI signal change can be extremely useful when comparisons are performed within the same voxels or ROIs under conditions that do not differ in the physical nature of the stimuli and responses involved in a given task. The use of extrinsic sensory stimulation to evoke signal changes has been a feature of most fMRI studies, and was crucial to the validation of fMRI’s ability to detect changes speciﬁcally related to localized information processing. The localization of primary sensory modalities in cortex is well known from human neuropsychology and animal experiments. For example, the occipital lobe is specialized for visual processing, whereas Heschl’s gyrus is similarly dedicated to auditory processing (Gazzaniga, 1999). Furthermore, visual processing related to determining the identity of objects is anatomically dissociable from that related to monitoring their spatial locations (Ungerleider and Haxby, 1994). FMRI has conﬁrmed and extended knowledge of the localization of primary sensory processing, and has even proven able to make ﬁne anatomical distinctions, such as the retinotopic mapping of multiple visual processing areas (Warnking et al., 2002) and the resolution of ocular dominance columns (Cheng et al., 2001). Although fMRI offers impressive spatial resolution in its detection of hemodynamic changes, considerable difﬁculties in interpretation of these changes arise when one moves beyond the primary sensory modalities.

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Since the neural bases of higher cognitive functions are not well characterized, problems arise when one attempts to use cognitive concepts to explain signal changes. In many neuroimaging experiments, task conditions are designed deliberately to manipulate the degree to which a theoretical cognitive process is involved in the subject’s performance. Brain areas that exhibit signal changes sensitive to these manipulations are then interpreted as playing a role in the cognitive process. Unfortunately, this necessitates circular reasoning, as there is little agreement among cognitive scientists about what independent cognitive subprocesses, or ‘modules’ (Fodor, 1983), exist. In primary sensory processing, the basic primitives are well understood: photons striking the retina trigger a well-known cascade of electrical and biochemical events in the case of vision, as do hair-cell displacements in audition and in some forms of touch (Kandel et al., 2000). Unfortunately, no such body of knowledge characterizes our understanding of attention, language, and memory. Not only do we not know exactly where they are in the brain, we do not know what they are. Therefore, two scientists with diverging theoretical stances may interpret the same fMRI data differently, based on a disagreement over what cognitive processes are involved in the experimental task. This problem leads to a disappointing inconsistency in neuroimaging studies purporting to examine the same function (see e.g. Poeppel, 1996; Shulman, 1996), and encourages meta-analyses of large numbers of studies to ﬁnd common ground (Cabeza and Nyberg, 2000). Despite these difﬁculties of interpretation, neuroimaging studies of long-term memory have conﬁrmed some existing theoretical concepts about the broad dissociations between multiple brain systems, which interact to give rise to the body of phenomena known collectively as memory. In part, this success is attributable to the fact that memory is expressed via a set of independent, quantiﬁable performance criteria, and need not be thought of as a theoretical cognitive process. Subjects can display motor learning through faster and more accurate performance of a motor task, priming through faster reaction times, and long-term declarative memory through successful recognition or recall of previously presented stimuli. In all of these cases, an independent variable is generated that can serve as a basis for analyzing signal changes. Neural signals can then be correlated with these observable effects of memory in order to infer the underlying mechanisms, freeing experimenters from the circular process of presupposing the extent to which a task engages mnemonic processing and then interpreting signal changes as indicative of such processing. Analyses of the relationships between observable behavioral results and signal changes have, as described below, conﬁrmed that the MTL participates in a particular form of long-term memory known as declarative memory, and is thereby distinguished from other brain structures that underlie different kinds of LTM. Beyond this broad categorization, studies have identiﬁed a number of factors related to behavioral outcomes that inﬂuence signal levels in the MTL. A primary goal of this review is to assess whether or not these factors can be considered to indicate the existence of unique cognitive processes localized to the MTL, specialized for promoting the formation and subsequent use of long-term declarative memories.

14.3. LONG-TERM MEMORY: BROAD ANATOMICAL DISSOCIATIONS The phrase ‘long-term memory’ refers to changes in the information encoded in a nervous system that persist for an extended period, from several minutes to decades. It is distinguished from ‘working memory’ (WM), which refers to short-term active maintenance of information needed to perform a task, after which the information may be lost, or perhaps encoded into long-term memory as well. Long-term memory is further divided by theorists into independent forms of memory. One such form is declarative memory, deﬁned as the ‘acquisition, retention, and retrieval of knowledge that can be consciously and intentionally recollected’ (Gabrieli, 1998). This deﬁnition is quite satisfactory in the study of humans, who can typically express the content of declarative memories verbally. Nonetheless, animals (at least mammals) are presumed to have an analogous system, although it must be tested indirectly. It is distinguished from nondeclarative, or implicit memory, deﬁned as ‘knowledge expressed through experience-induced changes in performance’

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(Gabrieli, 1998). This includes such diverse phenomena as motor skill learning, probabilistic classiﬁcation tasks (Knowlton et al., 1994), repetition priming, and conditioning, even though, as discussed below, these different abilities are supported to some extent by separate brain systems, and are further divided into additional subsystems by some theorists. The primary distinction between declarative and nondeclarative memory originates in the study of braindamaged humans. Damage to certain brain structures is known to result in amnesia, a selective deﬁcit in declarative memory with spared nondeclarative memory (Baddeley et al., 2002). Amnesia frequently consists of both an anterograde component (failure to acquire new memories for events occurring after the lesion) and a graded retrograde component (failure to recall memories acquired during a variable period of time leading up to the lesion). The co-occurrence of these two deﬁcits, along with a sparing of memories from the more distant past, has led scientists to propose that the brain structures implicated in amnesia are responsible for acquiring and retaining new memories for a limited time, but that memories are ultimately ‘consolidated’ in diffuse regions of the neocortex independently of these dedicated structures (Brown, 2002). The most famous case of amnesia is the patient known as H.M., who underwent an extensive bilateral resection of brain tissue including the medial temporal lobe (MTL), an operation intended for the relief of crippling epileptic seizures. Although amnesic symptoms may also result from damage to speciﬁc regions of the thalamus and hypothalamus, it is thought that these lesions mainly exert their effect through disruption of a distributed system that depends on output from the MTL (Aggleton and Brown, 1999). Thus, the MTL remains at the forefront of research into the basis of declarative memory. Animal studies have conﬁrmed the importance of the MTL for long-term memory, and have also characterized to some extent the unique contributions of its various substructures, including the hippocampal formation (hippocampus proper, dentate gyrus, and subiculum) and three adjacent neocortical areas: the entorhinal, perirhinal, and parahippocampal cortices (Squire, 1992; Eichenbaum et al., 1994). Although H.M. suffers from a very severe anterograde amnesia, he and other similar patients perform at normal levels on tests of nondeclarative memory. For instance, amnesics exhibit normal levels of skill acquisition through training and practice on a motor task, despite reporting no conscious recollection of ever having performed the task before (Yamashita, 1993). This suggests that nondeclarative memory is subserved by brain systems independent of those underlying declarative memory. A double dissociation between these abilities is established by complementary ﬁndings in the study of patients afﬂicted by diseases of the basal ganglia, including Huntington’s and Parkinson’s diseases. These patients sometimes show impaired acquisition of motor skills (depending on exactly which circuits are affected), with intact priming and declarative memory (Vakil and Herishanu-Naaman, 1998; Schmidtke et al., 2002). The ﬁndings of human neuropsychology regarding long-term memory have served to establish an anatomical dissociation between systems underlying implicit memory, including the basal ganglia, and systems underlying declarative memory, including the medial temporal lobe. Neuroimaging studies have largely conﬁrmed the contrasting anatomical bases of these two major forms of long-term memory. For instance, a recent review by Doyon et al. (2003) reveals a complex pattern of signal increases and decreases associated with improving performance on motor skill tasks. These changes are most prevalent in the motor cortices, the striatum, and the cerebellum, all structures that were already implicated in motor learning on the basis of lesion data. Notably, effects of motor skill learning, a form of implicit memory, were not found in the MTL structures thought to be critical for declarative memory. Studies of declarative memory have instead found effects primarily in the medial temporal lobe and in several subregions of the prefrontal cortex (see below), whereas studies of priming have found effects in a broad range of neocortical areas (Schacter and Buckner, 1998). Additionally, a series of fMRI studies has examined in detail a task thought to involve both declarative and implicit learning, demonstrating that activations in medial temporal and striatal structures are anticorrelated, reﬂecting competitive interactions between the two systems (Poldrack

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et al., 1999, 2001). Therefore, fMRI results are broadly compatible with the pre-existing accounts of the major subdivisions of LTM, and have served to focus the attention of declarative memory researchers on the medial temporal lobe and the prefrontal cortex.

14.4. MTL INVOLVEMENT IN DECLARATIVE MEMORY ENCODING We will now consider studies of declarative memory in more detail, in order to illustrate how fMRI has helped to further our understanding of the nature of the mnemonic processing that occurs in the MTL. In this review, we conﬁne our discussion to studies of episodic memory, the long-term retention of information stemming from individual experiences, as opposed to semantic memory, which pertains to general facts abstracted over many experiences and may be subserved by independent brain systems (Tulving and Markowitsch, 1998). Acquisition of semantic memories is, by deﬁnition, all but impossible to study within the time constraints of a neuroimaging experiment, but recall of semantic memories has been examined in comparison with episodic recall (Maguire et al., 2000; Wiggs et al., 1999). Episodic memory is well suited for study within the common paradigm of presenting visual stimuli in the context of a classiﬁcation task performed by the subject. Exposure to individual stimuli constitute individual episodes, which may be subsequently remembered or forgotten. When stimuli are presented with the intention of inducing the formation of new memory traces, tasks are considered to involve encoding. These are contrasted with retrieval tasks, in which stimuli are intended to reactivate a previously stored trace either for simple recognition of the stimulus, or for cued recall of some additional information associated with it. Encoding and retrieval are intuitively appealing as subprocesses describing the functions of the MTL, but whether they are truly dissociable and accurate depictions of what goes on in that structure must be judged on the basis of available data. They are a product of the methodology that must be used to study memory; in everyday life, experience does not come in discrete pieces. Even though it is undisputable that information is encountered for the ﬁrst time at some point, and used at some later point, it is possible that there are no speciﬁc neural processes that can be construed as speciﬁcally accomplishing these tasks. An analogy to the processes of reading from and writing to a disk drive may prove to be misleading. Below, we review the speciﬁc cases in which selective activation of the MTL has been detected, and consider to what extent the pattern of results support the reality of encoding and retrieval as isolable physiological events. Considering the critical role of intact MTL structures for episodic memory function, it was quite surprising that most early neuroimaging studies (using PET) did not report any medial temporal activation during encoding (Shallice et al., 1994; Buckner et al., 1995). MTL activation was detected in encoding non-verbal stimuli such as faces (Haxby et al., 1996) and spatial information (Maguire et al., 1996), but the failure to detect it using word stimuli was puzzling, given that the classical neuropsychological tests that established the importance of the MTL for episodic memory had relied heavily on word recall tasks (Baddeley et al., 2002). In contrast, nearly all studies of episodic memory reported signiﬁcant activations in prefrontal cortex (for reviews see Rugg et al., 2002; Yancey and Phelps, 2001; Desgranges et al., 1998), despite the fact that performance on the clinical tests is not particularly sensitive to frontal lobe damage (Shimamura, 1995). In considering the role of prefrontal regions in episodic memory, it is difﬁcult to distinguish activity related speciﬁcally to episodic encoding and retrieval from activity involved in ‘online’ processing of stimuli, i.e. perception and working memory. The list of theoretical cognitive processes dependent on the prefrontal cortex is long (Miller and Cohen, 2001), and one cannot discount its importance in the performance of episodic memory tasks. As most cognitive processes not dependent on forming new memories appear to be intact in amnesics, though, we focus here on elucidating the unique contributions of the MTL, with the acknowledgement that other brain areas are critically involved in performing the same tasks. Because researchers had every reason to expect that tasks involving encoding of words should produce hippocampal activation when compared to resting baselines, the absence of such results engendered much

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speculation. A number of explanations were proposed to account for the apparent insensitivity of conventional imaging paradigms to neural activity changes in the hippocampal system. These included technical concerns such as the increased susceptibility of the MTL region to certain MRI signal acquisition artifacts (although this would apply only to fMRI, not to PET), and also partial volume effects, which may be especially pronounced in the MTL because several highly differentiated structures are typically contained within small volumes, in comparison with neocortical areas. Purely biological factors were also considered. For example, it was suggested that the particular kind of neural information processing in the MTL that leads to memory formation may simply not be characterized by the overall increases in neuronal ﬁring that presumably drive BOLD signal changes in other brain areas (Cohen et al., 1999). This idea enjoys some empirical support from the electrophysiological literature. Neighboring neurons in sensory and motor cortex tend to have very similar ﬁring properties, leading to theoretical concepts such as columns and hypercolumns (Kandel et al., 2000). In the hippocampus, when cells with speciﬁc ﬁring properties have been discovered, such as ‘place cells,’ they tend not to form such tightly correlated clusters (Shen and McNaughton, 1996). Consequently, neural coding in the hippocampus is described as sparser and more distributed than in the cortex (Barnes et al., 1990). Additionally, it is possible that the encoding-related activity in the MTL is nearly always engaged to a comparable degree, rendering it unsuitable for subtractive analysis methods (Haxby et al., 1996; Eichenbaum, 2001). These differences between MTL structures and neocortex should not be completely dismissed in the light of recent reports of selective MTL activation related to a variety of experimental manipulations. In general, ﬁndings of selective MTL activation have not resulted from comparisons of deliberate encoding tasks with resting states, perhaps because baseline MTL activity during commonly used resting states is actually quite high compared with several alternative baseline tasks (Stark and Squire, 2001). Therefore, it is especially important in studies of the MTL to use constant task demands. This is commonly accomplished by presenting visual stimuli to subjects, under instructions to make binary decisions about them by means of a button press. Experimental manipulations may involve the nature of the stimuli, their relative novelty, the nature of the decision made about them, and post-hoc assessment of encoding success based on subsequent memory tests for the stimuli. These practices have succeeded in eliciting selective activations in the MTL, due to incidental factors that may or may not indicate the involvement of a unitary underlying encoding process.

14.5. FACTORS RELATED TO ENCODING Among the most robust ﬁndings is that presentation of novel stimuli, compared to repeated items, elicits greater activation in the MTL. This has been observed using scenes (Gabrieli et al., 1997; Stern et al., 1996; Constable et al., 2000), words (Kirchhoff et al., 2000), and faces (Stern et al., 1996) in deliberate or incidental encoding tasks. Another general ﬁnding that has withstood multiple replications is that presentation of meaningful stimuli, such as words and line drawings, produces greater MTL activation than presentation of stimuli without any identiﬁable meaning, such as false fonts and abstract geometric patterns (Kelley et al., 1998; Martin et al., 1997; Golby et al., 2001). While the selective activations described above result from different stimuli over constant task demands, positive results have also come from changing task demands with constant stimulus characteristics. One such manipulation is known as ‘levels of processing’ (Craik, 2002). A subject’s task is to make some sort of binary decision about the stimuli, not knowing that he or she is subsequently going to be tested for recognition of them. Tasks that involve identiﬁcation of a stimulus, and consideration of its known properties, are considered to involve ‘deeper’ processing than ‘shallow’ tasks that only require a judgment based on the obvious physical characteristics of the stimuli. Judging whether a printed word is a concrete noun or an abstract noun is an example of a deep task, whereas judging whether or not a word contains

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the letter ‘t’ is a shallow task. Because deeper processing results in higher rates of subsequent recognition of stimuli, levels of processing manipulations provide a convenient way of inﬂuencing the extent to which memory encoding takes place while maintaining the same physical task conditions. Neuroimaging experiments have demonstrated that deeper processing induces greater MTL activation for words (Wagner et al., 1998) and line drawings (Henke et al., 1997). While a general process of memory encoding in the MTL might be expected to manifest itself through effects of novelty, meaningfulness, and depth of processing, numerous alternative explanations may be proposed to account for the co-occurrence of these effects in a particular area. In each of these cases, the condition with greater MTL activity can be characterized as more arousing, engaging, or interesting. While such characteristics of experiences do correlate with their memorability, they certainly do not by themselves constitute proof of an underlying encoding process. Furthermore, as these ﬁndings are also characteristic of prefrontal areas (Lee et al., 2000), they do not help to specify the role of the MTL compared to the PFC, despite the dissociations seen in the clinical neuropsychology literature. Therefore, it is necessary to consider the relationship between these ﬁndings and other physiological evidence of memory encoding within the MTL. One of the most productive means of studying brain activity related to encoding has been made possible in fMRI only with the advent of event-related analysis procedures. Electroencephalographic studies of memory have demonstrated, using episodic encoding paradigms similar to those used in the fMRI studies discussed above, that stimuli that are subsequently recognized successfully by the subject elicit different ERPs than those that are forgotten (Rugg and Coles, 1995). This ‘subsequent memory’ procedure can be used to classify individual trials post-hoc in event-related fMRI, generating average hemodynamic response timecourses to remembered and forgotten stimuli. Wagner et al. (1998) reported that hemodynamic responses (i.e. increases from an average pre-stimulus baseline) were enhanced for subsequently remembered words in left prefrontal and parahippocampal regions. Brewer et al. (1998) reported a similar enhancement for complex scene pictures, in right prefrontal and bilateral parahippocampal regions. This pattern of lateralization in encoding is consistent with the other selective activations reported above: a number of studies have demonstrated that MTL selective activations for novelty (Kelley et al., 1998; Golby et al., 2001) and meaningfulness (Martin et al., 1997) tend to be left-lateralized for words, and progressively more bilateral as verbalizability of the stimuli decreases (reviewed by Martin, 1999). Material-speciﬁc lateralization of encoding effects is, of course, reminiscent of the general verbal/nonverbal division of labor between the two cerebral hemispheres. Several theories of hippocampal function, based on computational models (McClelland et al., 1995; Gluck and Myers, 2001), posit that the MTL facilitates the long-term updating and retention of information in domain-speciﬁc regions of neocortex through establishing associative links between its inputs, and that the MTL-speciﬁc mechanisms that accomplish this feat are rather general; they establish associations between their inputs regardless of their modality. From this perspective, one would expect that similar anatomical dissociations may be found in both left and right hippocampi, but that differences between the two would be limited to the processing asymmetry that characterizes the human brain: language-related processing in the left hemisphere, and visuo-spatial processing in the right. In the next section of this chapter, we discuss a number of dissociations that have been noted regarding the differential roles of individual structures of the MTL. In the current state of the literature, however, differences between the two hemispheres have indeed been limited to the verbalization-related asymmetry, consistent with accounts of domain-general encoding mechanisms inherent in hippocampal circuitry (O’Reilly and Norman, 2002). The ﬂexibility of these mechanisms has been uniquely demonstrated in a neuroimaging study of epilepsy patients with unilateral MTL damage. Patients with right-sided damage exhibited compensatory increases in left MTL activation for viewing novel versus repeated nonverbal stimuli, whereas patients with left-sided damage exhibited an analogous effect in the right MTL for verbalizable stimuli (Golby et al., 2002).

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14.6. REALITY OF ENCODING AS A COGNITIVE PROCESS So far, we have discussed four separate factors that have been found to correlate with increased BOLD signal in the MTL: novelty, meaningfulness, deeper processing, and subsequent memory. We now consider whether these factors can be considered as separate reﬂections of an underlying unity, a physiological process of memory trace formation. Ultimately, a complete understanding of memory formation will require integration of ﬁndings on the systems and molecular levels (see e.g. Squire, 2000; Kandel, 2001). Within the domain of noninvasive study in humans, we attempt here to determine whether or not the effects delineated above do represent a homogenous physiological event, uniﬁed spatially in its extent within the MTL, and uniﬁed temporally in its post-stimulus timecourse. This question has been addressed through studies building on the initial ﬁndings already described, using the techniques of fMRI, EEG, and intracranial recordings. An fMRI study by Otten et al. (2001) directly compared subsequent memory effects with depth-ofprocessing effects by performing the subsequent memory procedure with words presented in both a deep task (living versus nonliving classiﬁcation) and a shallow task (judging whether the ﬁrst and last letters are in alphabetical order). Regions in left anterior hippocampus and left ventral anterior frontal gyrus showed effects of both manipulations, as a subset of a larger group of areas showing the depth-of-processing effect. This ﬁnding suggested that the levels-of-processing manipulation caused greater engagement of neuronal processing in these two areas related to encoding. However, a subsequent experiment (Otten and Rugg, 2001b) did not support that conclusion. The use of a different shallow task (syllable counting) failed to produce subsequent memory effects in these regions, but did so instead in bilateral parietal and fusiform regions. As a follow-up, Otten and Rugg (2001a) conducted an ERP investigation of subsequent memory effects in the living/nonliving task and the alphabetical judgment task. Although both tasks produced a subsequent memory effect, the two effects were of opposite polarity, with quite different scalp distributions. These ﬁndings indicate that the effects of levels-of-processing manipulations depend on the precise nature of the tasks chosen, and therefore argue against the hypothesis that the deep/shallow distinction entails a unique underlying process related speciﬁcally to memory formation. The relationship between novelty effects and subsequent memory effects has turned out to be rather more robust. An fMRI study directly crossed the two manipulations (Kirchhoff et al., 2000) and found a number of novelty-sensitive activations in anatomically connected prefrontal and lateral temporal regions, the precise location of which varied with respect to the demands of the encoding task. Novelty effects were also seen in the MTL, mainly in the left hemisphere for word encoding, and bilaterally for pictures. Crucially, the prefrontal and medial temporal regions that were modulated by novelty on a given task were also modulated by subsequent memory, demonstrating a deep relation between these effects despite the fact that they occur in multiple parallel systems for encoding information under different task demands. However, these results do not suggest that the underlying mechanism is anything unique to the MTL. Underlying unity for novelty and subsequent memory effects in the human hippocampus were strongly demonstrated, with high temporal resolution, by an intracranial EEG experiment in epilepsy patients undergoing presurgical electrophysiological monitoring procedures (Friedman and Sutton (1987). All patients exhibited a bilateral anterior MTL negative ERP component, known as ‘AMTL-N400,’ which was stronger for novel compared to repeated stimuli. The magnitude of the difference was signiﬁcantly correlated with subsequent recognition performance for the stimuli, an effect that was left-lateralized for words and rightlateralized for pictures. These ﬁndings contrast with negative results of Van Petten and Senkfor (1996), and Musen and Treisman (1990), who found no subsequent memory effect in scalp ERPs for nonverbal abstract and geometrical visual stimuli, but did ﬁnd such effects with verbal materials. These ﬁndings suggest that the effects of meaningfulness can also be associated with the novelty and subsequent memory effects (but see Wagner et al., 1999, for discussion). This uniﬁcation is concordant with the theoretical views of hippocampal function outlined above: the hippocampus establishes associations between higherorder multimodal inputs, inputs that would be greatly enriched for stimuli matching pre-existing conceptual

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representations, compared with abstract patterns that evoke little higher-order processing beyond perception of physical features. So far we have not presented evidence indicating that selective MTL activity in encoding directly reﬂects incidental processing inducive of lasting memory traces, as opposed to deliberate effort to memorize stimuli, nor have we dissociated the meaning of the selective activation effects in MTL from those in prefrontal cortex. Both of these points were addressed in an elegant experiment by Reber et al. (2002a). Words and faces were presented to subjects along with a cue to either remember the item, or to forget it deliberately. There was a signiﬁcant effect of the cue on subsequent memory performance for words, but not for faces. Although subsequent memory effects for words were found in both prefrontal and MTL regions, those in prefrontal cortex were accounted for by the identity of the cue, and hence can be considered to reﬂect deliberate effort. The MTL effects, in contrast, were independent of the cue. This ﬁnding strengthens the conclusion, drawn from a decade of failures and successes to ﬁnd encoding-related activity in the MTL, that increases in MTL activation reﬂect neural processes leading to memory formation that are driven mainly by incidental factors correlated with the encoding of information into LTM, but not by deliberate effort to memorize information. Neural activity within the MTL does play a speciﬁc role in the encoding of memories, but this role cannot be adequately described without speciﬁcation of its interaction with other brain areas. What we do know is that the encoding-related processes leading to increased BOLD signal in the hippocampus are relatively automatic and independent of conscious manipulation, in comparison with the processing performed in prefrontal cortical regions. This distinction is completely consistent with the anatomical distinctions between these two areas. The hippocampal system is characterized by a stereotypical morphology that is extraordinarily uniform across all mammals, whereas the prefrontal cortex is one of the most greatly expanded and altered regions in the brains of humans compared with other mammals (Deacon, 1997). It is not surprising, therefore, that the functions of the hippocampus may not depend on the capacity for elaborate manipulation of information in working memory that is a characteristic of higher primates (Courtney et al., 1998).

14.7. ENCODING VERSUS RETRIEVAL Up until now, we have put off a discussion of the distinction between encoding and retrieval tasks. On single-item encoding and recognition tasks, ‘encoding’ refers to the moment a stimulus is ﬁrst encountered, during which a long-term memory trace may or may not be formed, whereas ‘successful retrieval’ refers to the moment when a previously presented stimulus is again shown, and the subject recognizes it as old (a ‘hit’). This is distinguished from ‘correct rejection’ of novel stimuli, and from two error outcomes: ‘false alarms’ (the subject wrongly indicates that a novel stimulus is old), and ‘misses’ (the subject wrongly indicates that an old item is novel). Assuming mostly accurate performance, successful versus unsuccessful retrieval was compared using PET by Nyberg et al. (1996), who presented alternating blocks of mostly new and mostly old stimuli. Greater MTL activation was detected for old stimuli, suggesting that the MTL is differentially involved in retrieval success. This contrasts with studies that have primarily implicated prefrontal regions as being involved in effort to recall information, but not speciﬁcally driven by success (Buckner et al., 1998a, 1998b). Although reports of selective activation in the MTL during successful retrieval are encouraging evidence that the same system may play a dual role in both encoding and retrieval, upon further analysis a serious problem of interpretation results. We have discussed extensively above, ﬁndings of selective activation of MTL to novel stimuli. These stimuli, if not recognized by a subject, can be expected to induce encodingrelated activity. Old stimuli, in contrast, may induce retrieval of the memory trace for them, but may also induce further encoding (Buckner et al., 2001), making it very difﬁcult to separate out the processes of encoding and retrieval in a recognition paradigm. Indeed, if novel stimuli induce an encoding-speciﬁc

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response, and old stimuli induce a retrieval-speciﬁc response in the same structures, then no contrast may be seen between the two conditions. It has been proposed that this conﬂict underlies the failure of many early retrieval studies to ﬁnd effects in the MTL (Stark and Squire, 2000). A number of strategies have been employed to overcome the confound between encoding and retrieval. Gabrieli et al. (1997) employed a cross-modal retrieval task with an event-related design, in which subjects encoded line drawings (unscanned) and later made recognition judgments (scanned) on written nouns that either did or did not match the drawings. Encoding novel versus repeated pictures were scanned separately. The two tasks showed peak activations in different areas: encoding pictures induced noveltyspeciﬁc activation in bilateral parahippocampal cortex, consistent with the subsequent memory effect in that area described by Brewer et al. (1998), whereas successful retrieval gave rise to activations in an anterior MTL region centered on the subiculum. This suggests that activity within the hippocampal formation related to retrieval success may be dissociable from the decreases in hemodynamic response associated with repeated presentation of single items. Further support for this hypothesis is provided by the study of Stark and Squire (2000), who report increased activity in the hippocampus for successfully recognized words and pictures, relative to novel foils, when the very same words and pictures were used in the study phase without the cross-modal manipulation used in Gabrieli et al. (1997). The difference in activation between targets and foils was exacerbated on a second retrieval test, during which both types of stimuli had been previously viewed in the ﬁrst retrieval test. With this manipulation, the novelty encoding effect was at least partially controlled for, allowing the experimenters to demonstrate that activity within the hippocampal formation can relate to success in recognition as well as to encoding of novel information. Below, we return to this issue of confounds inherent in dissociating encoding and retrieval, as it has been greatly clariﬁed by further anatomical and behavioral distinctions within the medial temporal lobe declarative memory system.

14.8. SUBDIVISIONS OF MTL AND LONG-TERM MEMORY: CORRESPONDENCES The neuroimaging studies discussed above have established that MTL activation is not easily modulated by performance of memory tasks relative to a baseline, possibly because the MTL is always engaged to a considerable degree in integrating input from the cortex. However, we have suggested that effects of novelty, encoding success, and retrieval success seen in studies with constant sensory stimulation parameters may reﬂect the differential engagement of an underlying physiological process with speciﬁc mnemonic functions, not directly related to conscious effort. In the following sections, we examine the extent to which these effects have localizing value as indicators of memory activity. After reviewing the internal anatomy of the medial temporal lobe, we ﬁrst consider some studies that have sought to localize effects of novelty encoding to speciﬁc substructures of the MTL using strict anatomical delineation techniques. Much more informative, however, are studies that have localized effects of novelty, encoding success, and retrieval success to the hippocampal formation or extrahippocampal structures on the basis of behavioral distinctions motivated by theoretical distinctions between multiple forms of declarative memory, which have arisen from human and animal studies outside the ﬁeld of neuroimaging. Although the presence of differential signal changes related to these factors may be only an indirect indicator of mnemonic function, the presence of such effects in speciﬁc structures under different conditions closely matches the roles assigned to those structures in contemporary cognitive neuroscience of memory. In the preceding sections, we have used the term ‘medial temporal lobe’ to refer to a set of structures known to be critically involved in declarative memory. In fact, such a simple anatomical description belies the complexity of the brain structures that make up this region of the brain. The individual structures of the MTL differ greatly from each other in cellular organization, extrinsic and intrinsic connection patterns,

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and neurophysiological characteristics (Shepherd, 1997), especially in comparison with the much larger neocortex, which displays a remarkably consistent organization throughout its extent. Given the importance of the hippocampus and associated structures in memory, an enormous amount of research effort has gone into studying the information processing that occurs there. Whereas most cortical areas are interconnected reciprocally, neural signals are largely passed through the hippocampus in a unidirectional loop. Reciprocal inputs from higher-order sensory areas converge on the parahippocampal and perirhinal cortices, as well as the entorhinal cortex, which also receives input from the former two areas. From the entorhinal cortex, a mainly unidirectional multisynaptic circuit proceeds through the dentate gyrus, areas CA3 and CA1 of the hippocampus proper, and the subiculum, which then projects back to the entorhinal cortex (Amaral, 1999). Detailed analysis of the connectivity in this circuit has led to the suggestion that the individual areas are arranged in a ‘hierarchy of associativity’ (Lavenex and Amaral, 2000), in which the ‘integration, complexity, or abstraction of information likely increases’ as one goes through the circuit outlined above. Furthermore, the output of the MTL system projects widely and reciprocally throughout the cortex, including primary sensory areas (Lavenex and Amaral, 2000). The anatomical facts, therefore, support the emerging theoretical conception of the MTL as a system that helps to establish long-term associations between inputs whose basic representations reside in the neocortex. Nonetheless, the individual substructures may have dissociable roles, especially considering their hierarchical organization. Severe declarative memory impairment in humans can result from hippocampal lesions of quite limited extent, such as the selective ischemia in area CA1 of patient R.B. (Zola-Morgan et al., 1986), perhaps the most famous amnesia patient after H.M. However, this does not rule out critical and distinct roles for extrahippocampal structures, since they project through the hippocampus in a multisynaptic loop that can be disrupted at any point. Comparative studies of amnesics (Rempel-Clower et al., 1996) combined with controlled lesion experiments in monkeys (Zola-Morgan et al., 1994) have indicated that memory impairment on a variety of tasks increases as more components of the MTL are damaged. Such a general ﬁnding is of course compatible both with models that treat the MTL as an undifferentiated system and with those that assign speciﬁc functions to its components. Several lines of animal research have focused on discovering which aspects of long-term memory are crucially dependent on the hippocampus proper as opposed to other MTL areas (Rudy and Sutherland, 1989; Eichenbaum et al., 1994). Although speciﬁc accounts vary, there is much agreement that the hippocampus proper is especially necessary for ‘(1) strong and persistent memories for events including their constituent elements and the relationships among them, and (2) a capacity to express memories ﬂexibly across a wide range of circumstances’ (Eichenbaum et al., 1994). Tasks that stress simple recognition of single items, such as the ‘delayed nonmatch to sample’ paradigm in monkey research, are sensitive to MTL lesions, but generally intact when the lesions are conﬁned to the hippocampus (Squire, 1992). Based on a variety of behavioral, neurophysiological, clinical, and genetic dissociations between the hippocampus and the extrahippocampal cortices (reviewed by Brown and Aggleton, 2001), theorists have proposed that a general dichotomy between these structures may correspond to the theoretical division of declarative memory into independent processes of ‘familiarity’ and ‘recall.’ According to theories that recognize this dissociation, recognition judgments of single items may be supported either by a rich, episodic memory of one’s prior experience with that item, including contextual details, or instead, only by a more vague feeling of familiarity, in which the subject knows that a stimulus has been encountered before, but does not recall associated details. The former, but not the latter, is believed to require an intact hippocampus (Tulving and Markowitsch, 1998; Aggleton and Brown, 1999). The hippocampus has been described as necessary for binding together multiple inputs in a relational context (Eichenbaum et al., 1994), for supporting the ﬂexible use of learned information in new contexts (O’Reilly and Rudy, 2001), and for recognizing conjunctions of objects rather than individual objects (Brown and Aggleton, 2001). In

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the imaging studies discussed so far, success in either encoding or retrieval has been deﬁned as the correct discrimination of previously viewed single items from novel ones. In principle, such a judgment could be accomplished by mechanisms of either familiarity or recall. In the next section, we describe neuroimaging studies that have used novelty and success effects as a proxy for mnemonic function, but have been able to differentiate the roles of hippocampal and extrahippocampal structures through behavioral paradigms that exploit the distinction between episodic recall and ‘mere’ familiarity.

14.9. IMAGING STUDIES OF EPISODIC RECALL VERSUS FAMILIARITY The tendency in neuroimaging studies to treat the MTL as a unitary structure derives more from technical limitations than from any theoretical rationale. It is very difﬁcult to achieve sufﬁcient spatial resolution to differentiate the substructures of the MTL. Although the adult human hippocampus is 4–4.5 cm in length (Duvernoy, 1998), its anatomically distinguishable substructures extend all the way along its longitudinal axis, wrapped around each other in a shape aptly described as a ‘jellyroll.’ As a result, many studies have reported MTL activations as taking place in the hippocampus or the parahippocampal area, without further differentiation. Fortunately, even this gross distinction is useful. As a starting point, we consider two studies that used a simple novelty-encoding paradigm, comparing the presentation of novel visual stimuli with repeated ones, with no further manipulation. Both of these studies used special techniques designed to maximize the resolution of functional activity in the MTL by constraining the analyses to speciﬁc regions of interest (ROIs) deﬁned by strict anatomical criteria, using co-registered high-resolution structural images for guidance in individual subjects. Zeineh et al. (2001) used a ‘ﬂat-mapping’ technique to deﬁne six different ROIs within the MTL, each of which may be described as hippocampal or parahippocampal. This study found sustained novelty-speciﬁc activation to presentation of complex scene pictures in two parahippocampal areas, whereas two hippocampal areas exhibited activation only in the later portions of the experiment. These results are consistent with the conclusion, derived from animal studies (Squire, 1992; Brown and Aggleton, 2001) that the role of the hippocampal formation in recognition memory for single items is limited compared to that of parahippocampal structures. Reber et al. (2002b) used a similar anatomically constrained analysis procedure to separate activity in anterior hippocampus, posterior hippocampus, entorhinal cortex, perirhinal cortex, and parahippocampal gyrus (the latter three being extrahippocampal cortex). Bilateral novelty-speciﬁc activity was reported in all regions for picture encoding, and in left posterior hippocampus and parahippocampal gyrus for word encoding. These two studies of anatomical speciﬁcity within MTL, taken together, suggest that novelty encoding may in fact engage the hippocampus, but they do not help much in differentiating the speciﬁc functions of the hippocampus from those of adjacent regions. One possible interpretation is that novelty encoding may involve systems responsible for familiarity as well as those that support recall. Therefore, a number of studies have attempted to distinguish, both at encoding and retrieval, neural activity speciﬁcally related to these two processes, while continuing to use novelty and success effects as the best available indicator of localized mnemonic function. One simple way to distinguish between recall and familiarity is to have subjects make a three-way subjective judgment, as to whether single stimuli are ‘new’ (unrecognized), ‘known’ (only familiar), or ‘remembered’ (the subject can recall the experience of encountering it before). Such a distinction is known to elicit different ERP magnitudes for known and remembered stimuli (Rugg and Yonelinas, 2003), but whether that difference reﬂects a quantitative or a qualitative dissociation is not established. Eldridge et al. (2000) demonstrated increased activation in the hippocampus for single-item recognition trials for which subjects made a subjective judgment of ‘remembered,’ compared with items that were merely familiar or those that were not recognized at all. While this study supports a speciﬁc role for the hippocampus

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in episodic retrieval, other studies indicate that hippocampal activation during encoding may speciﬁcally support the formation of episodic traces. Davachi et al. (2003) demonstrated that hippocampal activation during encoding of single words predicted a subject’s ability to subsequently indicate in which of two tasks he or she had encountered the item, but did not correlate with the ability to simply identify the item as old instead of new. In contrast, the perirhinal cortex did show such a subsequent-recognition effect for encoding activation. Other studies have demonstrated novelty (Sperling et al., 2001) and subsequent-recall effects (Meltzer and Constable, submitted ) in the hippocampus during encoding of paired associate items. During presentation of triplets of words (Davachi and Wagner 2002), hippocampal activation was greater for an encoding task emphasizing inter-item associations than for one emphasizing processing of individual items, whereas the opposite was true of entorhinal and parahippocampal cortices. Furthermore, a hippocampal subsequentrecall effect was found only for words encoded in the relational task. Montaldi et al. (cited in Mayes and Montaldi, 1999) found that hippocampal activation was associated with novel conjunctions between items, but not for single-item novelty, whereas varying the task instructions had no effect if the amount of encoding success was the same between tasks. Taken together, these results suggest that novelty and encoding success effects are reﬂective of the formation of episodic and relational memory traces in the hippocampus, in a manner analogous to the effects seen in extrahippocampal MTL structures during the encoding of single items for subsequent familiarity. Additional evidence for such a dissociation comes from studies that have used retrieval demands other than single-item recognition. In free recall, for instance, a subject is asked to name as many items as he or she can from a previously presented set. This is much more difﬁcult than discrimination of old versus new items, as familiarity alone is insufﬁcient to accomplish the task, and as such it requires an intact hippocampus (Aggleton and Brown, 1999). Consistent with the lesion data, Fernandez et al. (1998) found, in a parametrically analyzed block design study, that activation in the posterior hippocampus correlated with the number of words presented in a given block that were subsequently recalled following a period of distraction. Similarly, cued recall of paired associates requires relational processing beyond that necessary for familiarity judgments. Zeineh et al. (2003) found activation in subregions of the hippocampal formation during both encoding and cued recall of face – name associations. Meltzer and Constable (submitted ) found that bilateral hippocampal activation was speciﬁcally enhanced during cued recall on trials in which a paired associate word was correctly recalled, compared to trials in which the subject was unable to recall the paired associate. These fMRI studies demonstrate that, to the extent to which novelty, encoding success, and retrieval success effects can be considered as indicative of an underlying mnemonic process, effects in the hippocampal formation are speciﬁc to relational and episodic memory traces, whereas familiarity for single items are supported by an analogous process in extrahippocampal structures of the MTL. This distinction corresponds closely to the roles assigned to MTL structures by contemporary memory theorists on the basis of computational models and lesion data (Eichenbaum et al., 1994; O’Reilly and Rudy, 2001; Tulving and Markowitsch, 1998). Furthermore, the hierarchical relationship between single-item and relational processing is entirely consistent with the anatomy of the medial temporal lobe, as extrahippocampal inputs converge in the hippocampus in a feedforward synaptic loop (Lavanex and Amaral, 2000). Thus, anatomical distribution of novelty and success-related activations within the MTL corresponds to the individual structures known to be necessary for item familiarity and episodic memory, and can be taken as additional support for the hypothesis that such effects reﬂect neural activity related to the encoding and retrieval of long-term memories.

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14.10. SUMMARY The search for selective activation in the medial temporal lobe began with the hypothesis, based on solid clinical and electrophysiological evidence, that neural activity in the MTL was related to the formation and recall of long-term memory traces. Cognitive tasks were designed to engage the theoretical processes of encoding and retrieval, with the expectation of producing signal increases relative to a resting baseline. Because such comparisons did not reveal selective activation in the MTL, experimental paradigms evolved to exploit an array of more subtle factors that do inﬂuence the amount of neural activity in the MTL under fairly constant task conditions. As a result, the cognitive neuroscience of memory is now faced with a complex pattern of results to evaluate, while the physiological reality of encoding and retrieval as independent processes speciﬁc to the MTL has not been proven. In particular, novelty and subsequent memory effects display a number of common characteristics, including two different anatomical distributions: lateralization effects related to verbalizability, and a hierarchical organization of sensitivity, to single-item familiarity and to associative links, in different substructures of the MTL. The fact that task-induced retrieval of recent memory traces induces activations with similar distributional characteristics suggests that both encoding and retrieval might be results of activity in the same distributed neural circuits. Although the hippocampus and surrounding structures are undoubtedly essential for the encoding and retrieval of memories, it has been quite surprising that neuroimaging studies have highlighted its role in these functions only indirectly. The fact that comparisons between different cognitive tasks seldom elicit differential activation of the MTL, whereas incidental factors unrelated to deliberate effort often do, indicates that the reality of encoding and retrieval as independent cognitive processes is still uncertain. It is possible that the MTL is engaged in performing its memory-related functions at all times, and that the neural activity accomplishing these functions is not isolable through subtractions of different tasks. While mnemonic functions may be modulated by attention, emotion, and other factors, it is clear that the hierarchical design of cognitive tasks is not an effective means of modulating medial temporal activation levels. This situation is in contrast to neocortical higher-order association areas (including the prefrontal cortex), which typically do respond robustly to such experimental design paradigms, and also to primary sensory areas, which tend to respond to sensory stimulation regardless of cognitive task demands. These differences demonstrate that it is impossible to isolate the functions of all brain areas solely through the manipulation of performance demands. In the MTL, the existence of a physiological process for memory encoding and retrieval can only be inferred indirectly from a pattern of incidental factors, none of which seem to relate to deliberate conscious processing. This pattern constitutes a new framework within which future studies will attempt to elucidate the nature of the MTL’s unique contributions to human memory.

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15 Using fMRI to Study the Mind and Brain Bruce E. Wexler Department of Psychiatry, Yale University School of Medicine, MR Center, P.O. Box 208068, New Haven, CT 06520-8068, USA

15.1 Studies Assuming Localized and (often) Fixed Relationships between Cognitive and Brain Functions 15.1.1 Examples 15.1.2 Problems 15.2 Studies of Changing Relationships between Brain Function and Cognition 15.2.1 Changes in Activation with Task Repetition and Learning 15.2.2 Changes in Activation with Age 15.2.3 Changes in Activation with Changes in Physiological State 15.2.4 Effects of Shifting Attention on Activation 15.2.5 Impact of Brain Injury or Disease on Functional Activation 15.3 Summary

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In science our theories constrain both the way we conceive and the way we interpret experiments. The more limited our understanding of a ﬁeld of inquiry, the greater the gap between theory and reality, the more limiting are these constraints. Consequently, theories about how the brain works, even nascent and implicit, have major effects on how we design and interpret brain-imaging experiments. Brain-imaging research is immersed in the confusion of the mind–brain conundrum. Mind and brain are generally considered two sides of the same coin; moreover, we think, with some justiﬁcation, that we are chipping away at the mind–brain problem with each experiment. A major difﬁculty, however, is that there is no well-developed solution to the problem. Research often slips back and forth between mind and brain perspectives and loses focus on the question being addressed. Not infrequently we pose experimental Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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questions in psychological terms, collect and present data in brain terms, and do little more than wave hands when discussing what the experiment had to say about the initial questions. These are very difﬁcult problems. In this chapter, I will discuss them in the context provided by the intersection of two theoretical dimensions: (1) localizationist versus distributed models of brain function, and (2) ﬁxed versus changing relationships between brain structure and function. Localizationist models equate speciﬁc cognitive functions with speciﬁc brain locations; a particular cognitive operation is carried out by brain tissue at a particular location, and the functional property of that brain tissue is that particular cognitive operation. Distributed models, in contrast, postulate that even basic cognitive operations emerge from the integrated action of brain tissue at several locations, and that the functional properties of speciﬁc brain regions cannot be characterized as a cognitive operation. Models with ﬁxed relationships between brain structure and function assume that a particular cognitive operation is carried out by the brain in the same way at all times. Models with changing relationships between structure and function assume that a particular cognitive operation may be carried out by the brain in a different location, or by a different collection of regional activations, at different times. The time scale may be anywhere from seconds to years. The nature of the assumed relationship between structure and function in localizationist models often also leads to the assumption that the relationship between structure and cognitive function is ﬁxed or constant across time. I will provide examples of studies based on these different theoretical assumptions, and discuss some of their relative strengths and weaknesses.

15.1. STUDIES ASSUMING LOCALIZED AND (OFTEN) FIXED RELATIONSHIPS BETWEEN COGNITIVE AND BRAIN FUNCTIONS The great majority of early functional imaging studies, both with PET and with fMRI, were of this type, and essentially all established imaging programs include a number of such studies in their portfolios. These ‘where is it done’ studies are the easiest to conceive and analyze. It can be argued with some justiﬁcation that results of those studies provide a skeleton of cerebral functional organization upon which to hang the complexities of inter- and intra-personal variability. However, problems with this approach are signiﬁcant enough to raise the possibility that they are fundamentally misleading.

15.1.1. Examples In their 1988 Science paper ‘Localization of cognitive operations in the human brain’, Posner et al. (1988) articulated a guiding rationale for studies of ﬁxed, localized aspects of brain function, and described the ‘subtraction’ method central to their design. As they state, ‘The hypothesis is that elementary operations forming the basis of cognitive analysis of human tasks are strictly localized.’ They described earlier experiments in which subjects were asked to determine whether two uppercase (AA) letters were the same, whether an upper and a lower case letter (Aa) were the same, or whether two letters belonged to the came category (Ac). The dependent measure, reaction time, increased during this task progression. They suggested that the differences in reaction times indicated that different cognitive operations were involved in each task variant, and that each operation is related to a separate neural system. In their studies of language operations, they constructed a series of progressively more complex activation tasks. Each task served as a ‘control’ for the next more complex task. Activations associated with the ‘control’ task were subtracted from activations associated with the more complex task in order to identify the brain regions associated with the cognitive operations present only in the more complex task. For example, activations associated with viewing a simple ﬁxation cross were subtracted from activations associated with passively viewing a series of words. All areas showing signiﬁcantly greater activity during word viewing were in the occipital lobe. The authors ‘conclude that visual word forms are developed in the occipital lobe’. They suggested

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that particular areas are speciﬁc to visual words since they are activated neither by auditory words nor visual checkerboards nor dot patterns. They next subtracted the activations associated with passive viewing of words from those seen when subjects are asked to mentally note the names of dangerous animals in the series of visual words. Two areas survived this subtraction, one in the anterior left frontal lobe and one in the anterior cingulate gyrus. Based on results of other studies, the authors concluded that the left frontal activation is ‘speciﬁc to semantic language tasks’ while the cingulate activation is ‘identiﬁed’ with attention. Multiple studies, including many from our imaging center at Yale also fall into the category of assuming localized relationships between cognitive and brain function. In one, activations in English-only speaking subjects listening to sentences spoken in Turkish were subtracted from activations in the same subjects listening to the same sentences spoken by the same speaker in English (Schlosser et al., 1998). Activations related to listening to the English but not the Turkish sentences were found in the left posterior temporal and the left inferior frontal lobes. The authors ‘conclude that the activation pattern evoked by the English sentences reﬂects auditory comprehension.’ They note that another study found much more extensive activations associated with an auditory semantic judgment task when a baseline without auditory stimulation was used for subtraction, but ‘assert that the more local focal activations in the present study likely reﬂect the isolation of language comprehension from lower-level auditory processing.’ Other studies have compared: object and spatial memory tasks to each other and to perceptual tasks in order to identify speciﬁc areas associated with speciﬁc types of memory (McCarthy et al., 1996); orthographic, rhyming, and semantic matching tasks to identify regions associated with speciﬁc components of reading (Pugh et al., 1997); and re-reading and immediately recalling of words to identify regions associated with component stages of working memory (Raye et al.,2002). There are probably over a thousand reports in the literature of studies conceived and interpreted in this manner.

15.1.2. Problems 15.1.2.1. Thresholds for signiﬁcance The problem here is that there is no agreed-upon, and probably no fully satisfactory, way to select a probability threshold for statistical signiﬁcance of differences between experimental and baseline conditions. At a scientiﬁc meeting 8 years ago, a group of Russian scientists complained to me that Americans used too restrictive thresholds, thereby creating a false sense of very few and highly localized activation differences between experimental and baseline conditions. If less restrictive thresholds are used, the same experimental conditions appear to be associated with more widely distributed activations of multiple brain areas. Shulman and colleagues have looked at this issue experimentally in high ﬁeld imaging of rat olfactory cortex (Figure 15.1). If the signiﬁcance threshold is held constant but the concentration of odorant is increased (columns 1–3), there is a progressive increase in areas activated. Thus there seems to be a widely distributed potential response system that becomes more activated as stimulus intensity increases. When the activation with the lowest level of odorant is evaluated at a more relaxed threshold (column 4), more areas appear activated and the expanded activation image matches that seen with the highest concentration of odorant at the more restrictive threshold. In other words, when the threshold is relaxed, the newly appreciated activations appear to be meaningful (signal) rather than artifactual (noise). The Russians may have had a point! 15.1.2.2. Matching experimental and baseline tasks for difﬁculty and interest One of the most difﬁcult problems in choosing experimental and baseline tasks for a subtraction paradigm is matching them for difﬁculty and interest. Both increased task difﬁculty and increased interest of a task can increase task-associated brain activation. Thus, an experimental task may generate greater brain

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Figure 15.1. Evidence that activations evident at less restrictive thresholds are meaningful. The activation intensity increases with increasing odorant concentration (left three panels). However, when the pattern elicited at low concentration (left panel) is normalized to the pattern at high concentration by lowering the threshold for display, the topography of the patterns at different concentrations is similar (third and right panels). This suggests that the activations evident at the less restrictive thresholds (right panel) are meaningful and not noise.

activation than a baseline task because it is more interesting or more difﬁcult rather than because of the differences between experimental and baseline tasks designated by the researchers. In the Posner et al. study, for example, monitoring a list of words for dangerous animals may be more difﬁcult and more interesting than passive viewing of words, and passive viewing of words may be more interesting than looking at a ﬁxation cross. Hence, the experiment might be exploring the brain activations associated more generally with ‘interest’. McCarthy et al. have data on this issue. In an initial study, they reported memory-related activations in the middle frontal gyrus based on differences between their memory task and a perceptual baseline task (McCarthy et al., 1994). In a subsequent study they found that a more difﬁcult perceptual task produced a signiﬁcantly smaller difference signal than the original perceptual task in this same region. By subtraction in the ﬁrst study, the area was identiﬁed as a memory and not a perceptual area. However, in the second study, using a ‘parametric’ design (Friston et al., 1996) the same area could be identiﬁed as showing activity related to the difﬁculty of perception without a memory component. 15.1.2.3. Problems intrinsic to the subtraction concept itself Vygotsky, writing in 1960, summarized and critiqued the subtraction design as it had been applied in experimental psychology with reaction time as the dependent measure (Vygotsky, 1978). In those experiments, the simplest baseline condition consisted of having the subject respond to the appearance of a single, repeating stimulus (simple recognition reaction time, RS ). In the next more complex condition, subjects were required to discriminate between a speciﬁc target stimulus and other stimuli. Total reaction time (RT ) was then assumed to reﬂect RS + RD , with RD being the time required for discrimination. In some experiments, the situation was then further complicated by asking the subject to respond to stimulus A with their right hand and stimulus B with their left hand. RT was then assumed to consist of RS + RD + RC , with RC being the time required to choose the correct movement. As Vygotsky summarized the idea, ‘. . .the discrimination response is a simple reaction plus discrimination; the choice reaction is a simple reaction plus discrimination plus choice. The higher, more complex response is seen as the arithmetic sum of its elementary components.’ He cited studies that assumed that ‘by subtracting the time needed to comprehend and name a word from the time needed to comprehend, translate a word into a different language, and name it, we can obtain a pure measure of the translation process. In short, even higher processes such as speech comprehension and production can be analyzed by these methods. A more mechanical notion of the complex, higher forms of behavior would be hard to imagine. . . . In our view, the basic premise

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underlying this entire line of analysis is incorrect. It is not true that a complex reaction consists of a chain of separate processes which may be arbitrarily added and subtracted. Any such reaction reﬂects processes that depend upon the entire process of learning at every level of practice.’ Friston et al. (1996) offered a critique of cognitive subtraction designs in brain imaging conceptually similar to Vygotsky’s, and followed with an empirical demonstration of its limitations. They write that the subtraction ‘approach, predicated on a pure insertion, assumes that each cognitive component evokes an ‘extra’ physiological activation that is the same irrespective of the cognitive or physiological context.’ They ‘suggest that nonlinear systems like the brain do not behave in a fashion that is consistent with cognitive subtraction.’ They argue that factorial designs that allow for interaction effects provide more appropriate conceptual and experimental approaches. In an experimental demonstration, Friston et al. evaluated activations apparently associated with object recognition by comparing cognitive subtraction and factorial approaches. They hypothesized that inferotemporal activation associated with object recognition would change as a function of whether or not phonological reference was also required. If so, assumptions of additive linearity and pure insertion would be violated. To test this, Friston et al. ﬁrst created two different subtractions that should each isolate activity associated with object recognition. The ﬁrst compared activation when subjects simply said yes when presented with any colored shape to activation when they said yes only when the colored shape was an object (e.g., a tree) rather than an abstract form; only the latter condition required object recognition. The second subtraction compared activation when subjects named the color of a presented shape to activation when they named a visually presented colored object. In this contrast both conditions required phonological retrieval but only the latter required object recognition. As hypothesized, inferotemporal activation associated with object recognition was very different in the two cognitive contexts; it was almost 10-fold higher in the presence of concurrent phonological retrieval. Friston and colleagues then applied a factorial analysis to the four experimental conditions. This analysis identiﬁed a robust area of interaction between object recognition and phonological retrieval in the inferotemporal cortex as well as in the angular gyrus. They conclude that ‘pure insertion disallows any interactions and yet these interactions are evident, even in the simplest experiments.’ Identiﬁcation of the interactions still derives from a subtraction process, but by comparing incremental changes in activity across regions and conditions it identiﬁes changing interactive processes among neural and cognitive processes (see discussion below of studies of aspects of brain structure–function relationships.) It may be tempting to interpret the main effect of object recognition and phonological retrieval that are also identiﬁed in the factorial analyses as primary locations of these functions (i.e., areas active in association with these cognitive functions in both experimental contexts), but the general difﬁculty of assigning meaning to main effects in the presence of signiﬁcant interactions certainly applies here as well as in other types of research. At the least, it would require creating a large number of cognitive contexts in which to embed a particular function, i.e., many factors, to feel conﬁdent of the stability of the main effect. Finally, Shulman and colleagues have done a series of MRS, fMRI and single-cell electrical recordings which together make a powerful argument for the functional importance of what is subtracted away in subtraction paradigms, even for the speciﬁc cognitive activity putatively isolated by the subtraction. By quantitatively measuring rates of metabolism of glutamate to glutamine and rates of glucose oxidation, they determined that close to 75 % of brain energy consumption supports neurotransmission (see Chapter 5). By relating BOLD-derived changes in cerebral oxygen consumption to changes in electrical physiological measures of neuronal spiking, they demonstrated a stoichiometric relationship between the two that was in close agreement with the measured relationship between glutamate metabolism and glucose oxidation. Conservatively, they estimate that 75 % of brain oxygen metabolism is associated with neural activity. The problem this creates for the subtraction paradigm is that differences in the fMRI BOLD signal between the baseline and cognitive tasks are typically only a few percent of the energy. Since 75 % of brain oxygen consumption is linked to neuronal activity, this difference in bold signal represents a difference in neuronal

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activity only on the scale of approximately 2 %. If we march around the brain associating different cognitive operations each with 2 % of neuronal activity at different brain locations, what are we going to do with the unassigned majority of activity? And how are we going to study it with a paradigm that eliminates it? In accord with the experimental results described in the other chapters on brain activity, we argue that a 2 % increase in neuronal activity in an area is a marker that that area is associated in some way with the added task activity. However, the idea that absence of a small activity increase in an area means that it is not involved in the task is immediately problematic. This idea has no more a priori validity than saying that if a Beethoven symphony calls for 100 instruments, and a Brahms symphony those same 100 and three more, then the difference between the Brahms and the Beethoven is located exclusively in those three added instruments. Hyder et al. (Chapter 10) have also demonstrated experimentally that the large amount of neuronal activity eliminated by subtraction procedures is essential for task performance, at least for the particular case they studied of anesthetized rats. They measured oxygen consumption and neuronal ﬁring in the somatosensory area during forepaw stimulation of rats under varying depths of anesthesia. They reasoned that if, as the subtraction paradigms suggest, increments in oxygen consumption and neuronal ﬁring are sufﬁcient to support the somatosensory processing, then incremental changes following stimulation should be the same regardless of the depth of anesthesia. They found instead that both oxygen consumption and neuronal ﬁring reached approximately the same total level during stimulation no matter how deep the anesthesia and how low the baseline metabolic and ﬁring rates. When the baseline starting point was lower, the task-related increments were greater. Rather than ﬁnding ﬁxed increments across anesthesia levels, they found ﬁxed activity end points reached by increments that were greater with lower baseline states. Apparently, then, most or all activity in the region is necessary to support the somatosensory processing. This fact underlines the problems for the subtraction paradigm that arose from the earlier work on brain energetics by Shulman et al. First, if most or all of the neuronal activity in a region is needed when that region is functionally active, how can it be adequately studied if the experimental design subtracts away 95 % of the activity? It is a bit like trying to learn about dogs by looking only at how their tails wag. Second, if functional engagement of a brain region involves activity of all or most of the neurons in the region, how can we be at all conﬁdent that if a region is only 97 % active (i.e., fails to show a 3 % increase in activity) it is not engaged by the task? 15.1.2.4. What is localized, anyway? Perhaps the most fundamental question to be asked about localizionist models and studies is ‘what is localized?’. One way to appreciate this question is to do a computer literature search of functional imaging papers sorted by a particular brain region. Most research reports are organized around the activation task rather than the brain region or regions activated, as evidenced for example in paper titles (e.g., ‘fMRI study of working memory’). In discussing their ﬁndings, most researchers compare their results with those of other studies using similar or related activation tasks. This can create the impression that the areas activated are associated primarily with the particular activation task and others very similar to it. If, however, the literature is searched by brain region, it is quickly apparent that individual regions (albeit coarsely deﬁned) are associated with a quite wide-ranging collection of functions. The facts that researchers tend to sort themselves into groups based on type of activation task rather than particular brain regions, and tend not to discuss the work of other groups using different types of activation tasks, contribute further to the sense that regions are specialized for cognitive tasks. A.R. Luria drew two general conclusions from examination of soldiers with localized brain injuries incurred during World Wars I and II (Luria, 1973). First, even highly localized lesions affect many different behaviors and functions, thus seeming to ‘locate’ multiple functions in the same area. Second, lesions in different places interfere with the same apparent behavior or function, seeming to ‘locate’ the same behavior

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in multiple areas. Luria concluded that cognitive functions result from the integrated action of multiple brain areas. The deﬁning characteristic of those ‘cerebral functional systems’ was production of an invariant outcome by variable means. Moreover, not only were behaviors as simple as breathing or as complex as memory not located in a single region, the distributed network of regions that generated the behavior varied from time to time. This was not as holistic a view as offered by Lashley, because only some regions were potentially involved in any given task, and whereas a speciﬁc region could contribute to multiple functions, it would not contribute to all functions. However, if we accept Luria’s view, the types of cognitive function often studied with brain imaging are not located in a speciﬁc region, and cannot even be said to be located in a speciﬁc set of regions, since the regions that come together to create the function change from time to time. Friston and colleagues suggested a similar view: ‘Cognitive processes themselves may only express themselves at the level of interaction.’ In both these views, the cognitive function is an emergent property of the interaction among multiple brain regions, and does not reside in any one. These views, then, argue that functions like those often studied with brain imaging methods are not localized in speciﬁc areas of the brain. This does not imply that all brain areas are functionally equivalent. It says only that their functional differences do not correspond to speciﬁc cognitive functions. The question of how to characterize the functional characteristics of speciﬁc brain regions, the question of what is localized, remains open. Patricia Churchland develops this openness more fully in Neurophilosophy (Churchland, 1986). She suggests that new knowledge about the functional organization of the brain may lead to new ways to think about psychological operations. The current concepts of psychology, she argues, may eventually be seen as a ‘folk psychology’ and replaced by a true neuropsychology. The same argument can be made in regard to cognitive psychology, since despite its laboratory rigor it has little more of a neurological base than do the psychological concepts Churchland critiques. From this position, starting with concepts of cognitive psychology may be a misleading way to approach the task of studying brain organization and function, since there is little reason to believe that component cognitive processes as we have deﬁned them are homologous to component brain processes. In a 1985 article I discussed these issues by comparison of pictographic and alphabetic systems of writing (Wexler, 1986). In pictographic writing, each symbol represents a single thing, action, or idea. A thousand pictographs are needed to represent a thousand things, actions, or ideas. In an alphabetic language, individual symbols or letters do not have representational value; it is combinations of letters that represent things, actions, or ideas. Webster’s Dictionary has over 460 000 entries based on the 26 letters of the Roman alphabet! Words, the functional units of alphabetic languages, emerge from the combination of letters. This creates much greater functional capacity than is possible with a pictographic language. The functional organization of the brain, I suggested, is more like an alphabetic than a pictographic language; functionally differentiated regions serve as letters in a neuropsychological alphabet, and represent cognitive functions in combination but not in isolation. A cognitive localizationist view of brain function, on the other hand, is more like a pictographic language. The task, in order to move beyond the localizationist view that equates speciﬁc brain regions with speciﬁc cognitive operations, is to discover the letters of the brain’s alphabet and how they are combined into words and sentences that correspond to recognizable components of cognition. The analogy breaks down, however, and the complexity of the conceptual challenge increases, when we appreciate that the letters or components themselves can change when in combination and that the processes of combination can be nonlinear.

15.2. STUDIES OF CHANGING RELATIONSHIPS BETWEEN BRAIN FUNCTION AND COGNITION The limitations of simple subtraction/localizationist experiments have been increasingly appreciated by the imaging community, and newer approaches have focused on changing and distributed aspects of the

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relationship between brain function and cognition. Changes with time in the relationship between brain function and cognition have been studied with a variety of approaches. Studies have compared regional activation associated with the same experimental task when the task is simply repeated, when the subject is in different baseline physiological states, after practice and learning, in young and old subjects, and in people with and without the effects of neurologic illness or injury. Others have looked at the effects of other cognitive activity, preceding or concurrent with the index task, on activity associated with the index task. A third set of studies has demonstrated changes in activity as a function of the direction of attention. I will describe examples of studies focusing on each of these types of changes in the brain activation correlates of cognitive activity, and discuss what types of things we can learn and have learned from them about distributed as well as changing aspects of the relationship between cognitive activity and brain function. I will conclude by considering which of the problems identiﬁed in reference to static localizationist studies are also problematic in these studies.

15.2.1. Changes in Activation with Task Repetition and Learning In the most basic of these studies, subjects did the same simple repetitive motor movements during multiple imaging sessions. In one, the same subject was imaged on nine occasions over six weeks while tapping with his right index ﬁnger (Wexler et al., 1997). A device constructed for the study ensured that the force and range of motion were constant throughout. The rate of tapping was paced by an auditory signal and conﬁrmed by observation. A highly similar area of the left sensorimotor cortex was activated in all sessions. What was of interest was that on one occasion, but not others, activation was also evident in the supplementary motor area at an intensity comparable to that in the sensorimotor cortex, and on another day activity of similar intensity was evident in parietal cortex. Activity in all these areas was evident during more complex motor tasks such as tapping according to a patterned sequence of different ﬁnger movements, indicating that they are part of a distributed motor system. Even such a basic task as single ﬁnger tapping may be associated with different activation patterns in the same individual on different occasions. A second, similar study, looked at activation associated with opening and closing of the ﬁst paced at 1 Hz by an auditory cue, in a group of 12 subjects scanned in an initial session and then in two follow-up sessions, one 5 h later and another 49 days later (Loubinoux et al., 2001). When this simple task was repeated after 5 h, task-related activation was greater in the contralateral premotor cortex and in a small area of the cerebellar cortex, and lesser in the contralateral sensorimotor and supplementary motor areas, and the parietal cortex and posterior cingulate. Most of these changes were diminished at 49 day follow-up. In the contralateral sensorimotor area, for example, the area of activation was reduced nearly 20 % at 5 h retest and actually was greater than the original response at 49 day follow-up. So here again we see multiple distributed and signiﬁcant changes in task-related activation when a very simple motor task is performed a second time 5 h after having ﬁrst done the task. Studies like these leave us with the unsettling question of which activation pattern is the real one? Understanding these results is complicated by the fact that similar tasks are done repeatedly throughout one’s life. Despite this, though, reconﬁguration of activation after a single session of opening and closing one’s ﬁst is similar enough across subjects to show signiﬁcant group effects, and durable enough to last at least 5 h. One must wonder, though, whether the major change from session 1 to session 2 is accommodation to the setting and experience of fMRI itself. It is true that the motor task is always differenced from the baseline activity associated with the general fMRI experience, but as we have seen when discussing static localizationist studies, changes in this baseline activity can alter task-related increments. Indeed, if one’s goal was to identify changes in brain activation associated with increasing familiarity with the fMRI environment, one might see the resting period as the active or experimental condition, and use a simple motor task as a baseline condition in which brain activation is relatively constant. In this case, the difference

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in difference scores between sessions 1 and 2 would be seen as changes in activity patterns associated with resting quietly in the magnet. These considerations demonstrate the difﬁculty of interpreting studies of even simple changes in the relationship between cognitive functions and brain activation; the multiple degrees of freedom require systematic experimental manipulation of multiple components of the situation and tasks before conclusions can be formulated. Studies like these, though, demonstrate how richly dynamic a system we seek to understand, and in their aggregate hold the promise of revealing some principles that govern these dynamics. Other studies have done repeated imaging while subjects learned to improve their performance on difﬁcult motor tasks. In one, for example, the investigators compared regional cerebral blood ﬂow measured with positron emission tomography early in an initial session when errors were high to later in that same session when errors were low (Shadmehr and Holcomb, 1997). Values at each voxel were normalized for overall brain blood ﬂow, and these relative ﬂow values during a resting baseline were then subtracted from values during the motor task. Changes in these incremental difference scores were limited to blood ﬂow decreases in the sensorimotor cortex and putamen, suggesting less neuronal activity as performance became more efﬁcient. More extensive changes were noted when comparisons were made between the later part of the initial session and a follow-up session 5.5 h later, even though performance was not signiﬁcantly different at these two time points. At follow-up, blood ﬂow was greater in the left posterior parietal cortex and the left dorsal premotor cortex, and lesser in the left and right middle frontal gyri within the prefrontal cortex. The authors attribute these changes to the heretofore hidden process of consolidation of learning that stabilizes mnemonic representations. Although we don’t see the process actually in action, we get clues as to what areas of the brain are more and less involved after consolidation. Moreover, it appears that consolidation involves decreases in activity in some regions and increases elsewhere. Efforts have also been made to study changes in activation related to learning cognitive tasks. For example, in one study subjects pushed a button when they thought stimulus cards signiﬁed rain (Poldrack et al., 1999). They were not told which combination of cards signiﬁed rain, but ﬁgured this out based on feedback throughout the imaging session. For subtraction purposes, a control task consisted of simply indicating when two, rather than one or three stimulus cards were present. Voxels showing changes in signal over the course of the imaging session while doing the rain prediction task but not while doing the control task were assumed to show learning-related changes in activation. Activity increased with learning in the right inferior prefrontal cortex, right and left inferior parietal cortex, posterior cingulate and right insula. Learning-related decreases in activity were seen in the medial prefrontal cortex, orbitofrontal cortex, left dorsolateral prefrontal cortex, and the right occipital cortex. In other words, more cortical areas showed activation changes over the course of learning than not; only the temporal lobes seemed unchanged. As discussed in other chapters, it is reasonable to conclude that if activity in a region changes when a task is done repeatedly, or under different conditions, it is evidence that the region is somehow involved with the task, at least at one or the other times or under one or the other experimental condition. Results like those just cited demonstrate that many widely distributed brain areas are involved in even simple tasks in controlled laboratory settings. A hope of studies like these is that the experimental ﬁndings will reveal principles and processes of dynamic change in the brain, and provide a foundation for understanding the interaction and integration of the distributed areas of activity. The large number of brain areas in which apparent task-related activity changes over time or under different conditions, however, makes this a daunting task. Moreover, it is even more complex than it looks, since brain regions may function at times by changes in ﬁring patterns within and across neurons that do not lead to overall changes in regional metabolic activity, and because the anatomic resolution of our imaging methods may be insufﬁcient to resolve distinct functional components. As a result, the conclusions we are able to draw from studies like these, while a valuable beginning, remain very general. For example, the very competent team of investigators who conducted the aforementioned study of the weather prediction task began the

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discussion of their ﬁndings by stating ‘The present ﬁndings are consistent with a number of other recent studies suggesting that skill learning involves changes in which neural networks are involved in performance (Poldrack et al., 1999).’ Even conclusions like this, however, must be qualiﬁed by the fact that the absence of activity differences in a region between task and control conditions, or the absence of changes in the difference before and after learning, to do not prove that the regions is not active during task performance.

15.2.2. Changes in Activation with Age Changes with age take place too slowly to be measured in within-subject designs that test the same individuals repeatedly. Instead, groups of younger and older subjects are compared with the assumption that differences represent changes that occur within individuals over time. For example, covert word generation in response to letter cues has been compared in children (mean age 10.7 years) and young adults (mean age 28.7 years) (Gaillard et al., 2000), and semantic encoding studied in young adults (mean age 25 years) and elderly adults (mean age 77 years) (Stebbins et al., 2002). Task performance was not monitored during imaging in the ﬁrst study, but in the second, younger and older subjects performed at similar levels. In both studies, activation was apparent in the inferior frontal cortex (Brodmann areas 44/45), the middle frontal gyrus (BA 46) and the superior frontal gyrus, facilitating comparison across studies. Children showed signiﬁcantly more extensive activation overall than young adults, with the differences greatest in the right hemisphere. While the activation task produced greater left than right hemisphere activation in nearly all subjects, this laterality was decreased in the children due to the more extensive right hemisphere activation. PET studies indicate that resting glucose metabolism and cerebral blood ﬂow in the frontal lobes are more than 50 % higher in 8- to 12-year-old children than in adults (Chugani et al., 1987; Chiron et al., 1992). Therefore, greater task-related increases in activity in children can not be explained by lower baseline levels. Moreover, the lateralized nature of the differences between children and young adults also makes a general metabolic explanation less likely. Older adults also show less strongly lateralized activity than young adults, but here the primary effect is lesser left hemisphere activation rather than greater right hemisphere activation, as seen in children. The extent of left hemisphere activation was signiﬁcantly greater in the younger than the older adults in the inferior, middle and superior frontal gyri while intergroup differences in corresponding right hemisphere structures did not approach signiﬁcance. Here again, the laterality of the group differences, together with the fact that older adults showed marginally greater activation than younger adults in the cingulate, make it unlikely that the ﬁndings are due to a general metabolic or vascular effect of aging. In this particular study, however, activation during a control task in which subjects decided whether letters were upper or lower case, was subtracted from activation during the semantic encoding task. This leaves open the possibility that older subjects actually had greater left hemisphere activation than younger subjects on the control task, rather than lesser left hemisphere activation on the experimental task. Despite this problem, it seems that a number of things can be learned from studies like these. First, here is still more evidence that the brain can do the same cognitive task with varying conﬁgurations of neural resources. Beyond this, some things are suggested about functional development and aging of the brain. The adult pattern of functional lateralization for language-related tasks is not yet developed in 10- to 12-year-old children. Children appear to activate their brains more extensively than young adults. This is similar, at least in a superﬁcial way, to the fact that activation is often more extensive when young adults do a task for the ﬁrst time than when they repeat it. With aging, lateralization of functional activation is altered again, this time perhaps with loss of the selective left hemisphere activation during language-related tasks. These observations may add to our understanding of normal neural development and aging when considered along with other types of information. Experimental paradigms like these may also be useful in studying abnormal development and aging.

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15.2.3. Changes in Activation with Changes in Physiological State Here the same activation task is given to the same individuals when they are in different general physiological states. The degree of hunger and the degree of sleep deprivation provide examples. It is common sense that when one is hungry, food-related stimuli become more salient. Imaging studies, though, can identify some of the ways that neural correlates of information processing change in association with this increased salience. In one study the signal associated with looking at pictures of tools was subtracted from signals associated with looking at pictures of food (LeBar et al., 2001). One group of subjects was scanned 8 h after having last eaten and then again 1 h later with a different stimulus set. A second group was scanned twice, each time just after having eaten, to control for retesting effects. The double subtraction of food items – tools in the hungry versus satiated state identiﬁed many regions that showed a greater response to food than to tools in the hungry than in the satiated state: the amygdala, parahippocampal gyrus, and anterior fusiform gyrus. Given that the physiological manipulation is known to increase perceptual and subjective sensitivity to food stimuli, it is reasonable to conclude that the ﬁndings are due to changes in the neural response to food stimuli, rather than to changes in the neural response to tools. It is not as clear, however, whether it is more useful to think that food-related activation decreased in these areas during the satiated state or increased in these areas during the hungry state. A paradigm like this might be useful in studying individuals with eating disorders, and evaluating treatments for these problems, but it is less clear what has been learned about the brain, or what the next step would be in this investigation of normal function. In a study of sleep deprivation, subjects were scanned after 30 h without sleep or after a normal night’s sleep (Drummond et al., 2000). During the experimental task, subjects read and attempted to memorize 20 words presented in blocks of ﬁve words. Between blocks, subjects did a control or baseline task in which they were asked to determine whether words were in upper or lower case letters. In the double subtraction of memory minus control task and sleep-deprived versus rested states, multiple changes were noted. Memory-related activation present during the rested state in the left prefrontal cortex increased following sleep deprivation while that in the temporal cortex decreased. Additional activations were noted following sleep deprivation in the parietal lobes bilaterally, in the right inferior frontal gyrus, and in the left middle frontal gyrus. Despite, or perhaps because of, these generally increased activations, performance was worse in the sleep-deprived condition. The increased activations might be seen as the greater effort one must make to do something when fatigued, effort to compensate for dysfunction or inefﬁciency in processing systems. Alternately, the greater activations might be the problem itself; an unruly and therefore a less focused and a less efﬁcient brain. Again we see evidence that they brain performs the same task in quite different ways at different times. The complex relationships between activity and performance are also evident; both increased and decreased activity can apparently be seen with either increased or decreased performance. And then there is the issue that we are never either fully rested or fully fatigued, but are often somewhere in between.

15.2.4. Effects of Shifting Attention on Activation The ability of self-directed attention to increase activations related to sensory processing has been demonstrated repeatedly. For example, in one study we presented auditory and visual stimuli to subjects simultaneously, with instructions to attend to the visual stimuli at some times and to the auditory stimuli at other times. Although the visual and auditory stimuli were received by peripheral sensory receptors throughout the experiment, voluntarily directing attention to one or the other increased the degree of activation in the respective sensory receptive areas. A recent study was able to evaluate this attentional ampliﬁcation further by having subjects attend selectively to one visual ﬁeld that contained upward and downward moving dots (Saenz et al., 2002). They were then instructed to alternately track either the upward or downward stream.

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All dots in the unattended visual ﬁeld moved in the same direction. Activation to the dots in the unattended visual ﬁeld (i.e., in the contralateral visual cortex) increased when subjects were tracking the stream in the attended ﬁeld that moved in the same direction as the dots in the unattended ﬁeld. Here, then, was evidence that this attentional modulation of brain response to a stimulus feature extended throughout visual processing systems in both hemispheres and to aspects of the visual ﬁeld to which conscious attention itself was not directed. This may seem only a small gain in knowledge about the brain and mind, but it is steps like these that our current methods allow. Another set of studies has examined changes in activation that result when subjects do and do not attend to simple motor tasks. These are in effect dual task experiments as described earlier by Kinsbourne and Cook (1971), since in the conditions when subjects are not attending to the motor task they are instead attending to a second task. In one study, for example, subjects moved their right index ﬁnger back and forth in the form of a ‘U’ at a constant frequency paced by a metronome (Binkofski et al., 2002). In one condition they looked at a screen on which green ﬂashes appeared intermittently but were told to attend only to their ﬁnger movements. In a second condition they were asked to attend to the green ﬂashes while moving their ﬁnger, and in a third to count the ﬂashes while moving their ﬁnger. For a baseline condition, they were asked to look at the screen and ﬂashes but not to move their ﬁnger. One area of the left motor cortex showed the same activation relative to the baseline in all three conditions. A second nonoverlapping area showed greater activation when attention was directed to the movement itself. When the motor attention condition was compared directly to the two visual attention conditions, differences associated with the direction of attention were evident in prefrontal cortex, premotor, superior and posterior parietal, extrastriatal, fusiform, precuneate and primary visual cortices. In a similar study, the motor task was to push a button with the right index ﬁnger at 1 Hz paced by a ﬂashing green light (Indovina and Sanes,2001). In the visual attention-low movement condition, subjects pushed the button in response to a blue ﬂash at 0.2 Hz but not in response to a red ﬂash at 1 Hz. In the movement and attention condition, subjects again pushed one button when the green light ﬂashed at 1 Hz, and another when the blue light ﬂashed at 0.2 Hz. For a baseline, subjects viewed a red cross ﬂashing at 1 Hz. The authors introduce their study with an explicit discussion of distributed and changing aspects of brain function. They saw themselves as testing the rather general hypotheses ‘that visual attention performed concurrently with a simple repetitive ﬁnger movement would reveal novel brain representations.’ The results indicated complex interactions. When the attention task was added to the movement task, there was an increase in activity in the left superior parietal lobule and in the precuneus. However, when the attention task alone was compared to the baseline, activity in these same areas was decreased. In addition, when the attention task was added to the movement task, there were also decreases in activity in the left insula and right fusiform areas. These same areas showed increases in activity when the attention task itself was compared to the baseline. Overall, the movement plus attention condition, when compared to the baseline, showed multiple activations not apparent (or below threshold) in either the movement versus baseline or attention versus baseline comparisons. These included the superior and inferior parietal lobules and the orbitofrontal cortex. The authors drew three very general conclusions from the data. First, the movement and attention condition ‘activated a widespread brain network.’ Second, ‘collectively these data indicate that visual attention during movement modiﬁes movement-related activation patterns in the human brain.’ And third, ‘attention can suppress or facilitate movement-related activation.’ These two studies illustrate changes in brain activation when the same task is performed in a different overall functional context. Interpretation is confounded, however, by some of the difﬁculties noted in discussion of the static – localizationist studies. Even interaction analyses rest on a subtraction foundation. Post-hoc efforts to understand interactions have to deal with the arbitrariness of thresholds. Combining tasks (e.g., motor and visual attention) raises issues of overall task difﬁculty, and requires comparison to single tasks of greater difﬁculty. But perhaps most problematic are the wide-ranging changes in activation

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in multiple brain regions from relatively simple experimental manipulations. Nobody seems able to do any more than catalog the results. At this point, the complexity is overwhelming rather than informative.

15.2.5. Impact of Brain Injury or Disease on Functional Activation Finally, I will mention in passing the now thousands of functional imaging studies of patients with a wide variety of brain injuries or illnesses. While these studies are primarily for the purpose of learning more about the clinical condition, the way the brain functions when compromised by injury or disease can potentially provide additional information about principles and processes of normal function. Like the studies of aging, these studies compare different groups of subjects rather than comparing the same individuals under different conditions. The cross subject comparisons, however, serve as a standin for comparing individuals before and after the onset of illness, and thus provide a wealth of information about possibilities and principles of changing relationships between cognitive function and brain activation. Here again the number of clinical conditions studied and the extent and variety of reported changes in brain activation, overwhelms current ability to interpret and integrate the observations. But here again there are also tantalizing bits of information that suggest the potential of this information to increase our understanding of the brain and mind. I will describe brieﬂy several of the most interesting of these bits of information. Following traumatic brain injury, patients showed right lateralized activation asymmetries on a task that healthy controls showed left lateralized activity (Christodoulo et al., 2001), and generally showed more widely distributed activity than did the healthy subjects (McAllister et al., 1999; Christodoulo et al., 2001). Like elderly adults, these brain-injured young adults lost the normal laterality of activation that develops between childhood and adulthood. Several lines of imaging data thus converge in pointing to the importance of laterality of functional organization. The data from brain injured patients suggests that focal gray matter lesions and/or the disruption of white matter tracts interferes with maintenance of this higher-order lateralized functional organization. Patients with HIV infection show somewhat different results, preserving the general pattern of activation seen in healthy subjects, but showing more extensive activation despite slower task performance (Chang et al., 2001). This is similar to the load-related increases in activation seen in healthy subjects when task difﬁculty is increased. Perhaps things are more difﬁcult for the HIV-infected brain. If so, knowledge of the brain pathology associated with HIV infection may shed light on aspects of normal function. Studies of patients recovering from strokes that affect motor function have demonstrated that components of the normal, distributed motor system that are unaffected by the stroke become more active as patients recover movement. In one sense this is an unsurprising recovery mechanism, but in another it reveals a functional interchangeability among components that is contrary to the assumption that each of these distinct but interconnected regions must have their own special functional contribution even if we have not yet ﬁgured out the functional alphabet. Finally, a whole series of studies in psychiatric patients, as well as some in normal aging and other illnesses, have found that patients or older individuals activate the same regions as do healthy or younger subjects but to a lesser degree. This is particularly interesting because the patients and older individuals often show this underactivation despite performing as well as they healthy and younger subjects on the activation task. What does it mean that patients do the tasks as well as controls but with less brain activation? Perhaps the brains of healthy subjects activate more than is necessary to do the task. This ‘elaborative activation’ could be an important aspect of normal brain function that is lost in illness (Wexler, 1991).

15.3. SUMMARY Functional imaging studies have now established beyond question that cognitive and motor functions depend upon the integrated action of many regions distributed throughout the brain. Moreover, the particular

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constellation of regions involved in any particular function changes throughout the life span, after simple repetition of the task, and after systematic learning and practice. The activation of speciﬁc components in these distributed functional systems is responsive to the direction of attention, as well as to the physiological and cognitive context in which the particular task is being executed. Together these observations indicate that the simple concepts and experiments that sought to determine the location of cognitive functions, and which were the foundation for most early functional imaging studies, have limited value. Questions remain as to how to deﬁne the characteristics and boundaries of functionally distinct brain regions, because all of the brain is not the same, how these regional components are integrated, and to what degree even these structure–function associations are stable. Imaging studies of the distributed aspects of brain function, and of the ways in which the relationship between cognitive function and brain activation changes, have the potential to continue providing new information about how the mind and brain work. However, serious difﬁculties continue to limit such studies. One is the fact that with fMRI, in many ways the most versatile of imaging methods, signals from two conditions must be compared which necessitates a subtraction of one from the other thereby hiding approximately 90 % of neuronal activity from view. Still, important aspects of brain function have been revealed. For example things that we think about as psychological or cognitive functions such as attention, perception or memory emerge from the interaction among brain regions, and different conﬁgurations of regional activation can support the same function, functional synonyms in the brain functional alphabet. The changes in functional activation patterns, however, are routinely so extensive as to overwhelm our current ability to understand the observations we make. There is great need for new concepts to integrate our ﬁndings and guide future work. Generative, nonlinear and combinatorial models may prove useful in this regard (Friston, 2002), but problems related to functional subtraction, threshold setting and task difﬁculty still persist.

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16 Brain and Mind: an NMR Perspective Robert G. Shulman Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

Fahmeed Hyder Department of Diagnostic Radiology, Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

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‘The fascination of a growing science lies in the work of the pioneers at the very borderland of the unknown, but to reach this frontier one must pass over well traveled roads; of these one of the safest and surest is the broad highway of thermodynamics.’ Lewis, G.N. and Randall, M. (1923) Thermodynamics and the Free Energy of Chemical Substances, McGraw-Hill, New York and London. ‘physiology traces . . . the results of mental processes which do not fall within the sphere of consciousness, and must therefore have remained inaccessible to us’ Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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Herman von Helmholtz. Academical Discourse at Heidelberg, November 22, 1862, published in Science and Culture: Popular and Philosophical Essays (1995) Ed. David Cahan, University of Chicago Press, Chicago, IL, p. 95. Neuroscience generally consists of bottom-up approaches that seek scientiﬁc answers to larger questions of brain function. However, upon a close examination of the scientiﬁc directions that are lumped together as neuroscience considerable diversity becomes apparent. Francis Crick proposed that an understanding of brain–mind can be found in terms of the constituent molecules (Crick, 1991). Although this proposal would be accepted by many neuroscientists it is not obvious that this commonality overcomes the differences between the subﬁelds of neuroscience. Very different questions are asked about molecules and cells in the studies of cellular axonal connections, genetic development and neurotransmission to mention just some of the many faces of neuroscience. Different methods are used and more importantly, different assumptions, with different worldviews, form these investigations’ starting points in the world of neuroscience. In the physical sciences understanding is based upon micro explanations of macro phenomena, which forms the generalized basis of Crick’s proposal. The systematic understanding reached by atomic physics, the physics of matter, represents for many the scientiﬁc paradigm. Its micro explanations of macro phenomena have created our technological world and it has given us control of many natural forces. To ‘understand’ heat is to know how it depends upon the energies of particles and while water has long been known as H2 O such understanding is improved by quantum and statistical explanations. The goal of understanding complex matter, which is particularly acute for living organisms, brings into question the most reliable and informative ways to generate these explanations. Varied criteria are chosen as the starting points for different physical explanations of brain matter. The choices made of these criteria, of the starting assumption as to what the problem is and what the answer might be, distinguishes the various scientiﬁc disciplines lumped together as neuroscience. The hopes of neuroscience derive from the bottom-up successes of physics which is the study of energy and matter. When applied to the molecules and structures of life which exist in aqueous solutions, physics is most directly formulated as thermodynamics. The laws of classical, nonquantum physics as applied to the solutions of molecules found in biology are codiﬁed as Gibbs free energy, which is the ability to do work. This explanatory level is intermediate between the too diffuse, as for example laminar and turbulent ﬂow would be, or too microscopic, as it would be if we started from the fundamental particles. The success of thermodynamics in explaining the work of skeletal muscle, a functional organ of life, in which energy consumption and work done have been clearly deﬁned, exempliﬁes the achievements of this level of explanation in biological science. Neuroscience research described in this book has been based upon experimentally relating brain energy consumption (in the form of oxygen usage) to brain work done (in the form of neuronal ﬁring). From the relations established between brain energy consumption and neuronal activity, we have addressed the more complex questions of brain function. This approach is a unique starting point for bottom-up studies because for modern science it is the ultimate bottom. A work–energy relationship is more directly based upon physics than are the molecular, genetic, or psychological assumptions postulated in other subﬁelds of neuroscience. Although thermodynamical explorations of brain function may at ﬁrst seem unacceptably distant from the qualities of mind which form the ultimate goals of neuroscientiﬁc enquiry, we propose to show that, because of their reliability, they form a particularly valuable methodology. Further by starting with these thermodynamical assumptions we are unequivocally considering the brain to be subject to the laws of physics concerning energy–work. Our assumptions are that the brain–mind in this respect is not different from any other material, whose thermodynamical properties have been elucidated in the past four centuries by empirical physical studies of matter. By adopting an empirical research program based upon this intrinsic view of brain, we hope that ﬁrmly based physical studies of the brain’s organic matter will lead to a novel vantage point for understanding

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its more complex properties. We do not undervalue the qualities of mind that are the essence of human life. On the contrary we think they are too important to be assumed in order to study the brain. Qualities of mind are the ultimate, although, in our opinion, the distant goals of brain studies. Our approach differs from many neuroscientiﬁc studies which, starting from a presumed understanding of how the brain works, strive to understand how the brain subserves these assumptions.

16.1.

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Nuclear magnetic resonance studies of the brain have delineated the coupling between brain energy consumption and work. Magnetic resonance spectroscopy (MRS) has non-invasively measured in vivo concentrations and kinetics of molecular metabolites. These small molecules, e.g. glutamate, glutamine, lactate, glucose, etc. are studied as the components of the energetic and neurotransmitter pathways (for reviews see Shulman et al., 2002, 2003; Rothman et al., 1999). The understanding that glucose oxidation provides the brain energy was continued by 13 C MRS measurements of the ﬂux from labeled substrates like 1-13 C glucose through the tricarboxylic acid (TCA) cycle. Numerical values of the neuronal metabolic rate of oxygen consumption obtained from this measure of TCA cycle ﬂux agreed with measurements by other methods described in Chapter 2. The second thermodynamic measurement was of the neuronal work done by the brain. This work is coupled to the release of the neurotransmitter glutamate upon neuronal ﬁring and its subsequent recycling through glutamine (Figure 16.1). Glutamate is the major cortical neurotransmitter serving that function in Presynaptic neuron

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Figure 16.1. A model of glutamate–glutamine neurotransmitter cycling between neurons and astrocytes (for a review see Magistretti et al., 1999). The rate of the glutamate-to-glutamine cycle (Vcyc ) has been quantitated by 13 C MRS (for reviews see Rothman et al. 1999, 2002). Action potentials or spikes (shown by curved arrows) reach the pre-synaptic terminal to initiate the release of vesicular glutamate into the synaptic cleft. This activates the glutamate receptors in the post-synaptic neuron, which is a step required in the propagation of spiking activity to the adjacent neuron (in an all-or-none manner). The extracellular glutamate is removed rapidly by Na+ -coupled transport into astrocytes, where it is converted into glutamine. The synthesized glutamine then passively diffuses back to the neuron, and after re-conversion to glutamate, is repackaged into vesicles. (Modiﬁed from Shulman and Rothman, 2001).

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∼80 % of the neurons. The ability of 13 C NMR to follow the time course of 13 C label ﬂow into glutamate and subsequently into glutamine in the human brain has opened glutamate neurotransmitter cycling to quantitation. Soon after these ﬁrst experiments on human subjects, the easily manipulated rat model was exploited to expand the understanding of the interactions of the different rates in vivo (for a review see Rothman et al., 1999). The relative rates of neuronal oxidation (CMRN glu(ox) ) and glutamate cycling (Vcyc ) were measured over a wide range anesthesis in the rat (Sibson et al., 1998; Chapter 5). Simultaneous measurements of the 13 C ﬂow into glutamate and its subsequent ﬂow into glutamine were converted into CMRN glu(ox) and Vcyc , respectively. Figure 16.2 and Equation (16.1) summarize the results CMRN glu(ox) = Vcyc + 0.1

(16.1)

in units of µmol/g/ min. The results are signiﬁcant for understanding brain activity in several respects. First, a quantitative relationship between neurotransmission (i.e., neurotransmitter ﬂux) and neuroenergetics (i.e., neuronal oxidation) quantiﬁed the model in Figure 16.1. Second, at the intercept of Figure 16.2 as Vcyc approached zero, which corresponds to no neuronal ﬁring, CMRN glu(ox) falls to 20 % of the value at the nonstimulated awake condition. Hence in the nonstimulated awake condition ∼80 % of the energy supports events associated with neurotransmission. Third the slope of the CMRN glu(ox) and Vcyc curve is ∼1 (Figure 16.2 and Equation (16.1)), hence for every additional glucose molecule oxidized neuranally, one glutamate is released as a neurotransmitter and cycled through glutamine. This stoichiometry allows changes in glutamate neurotransmitter ﬂux to be determined from measured changes in oxygen consumption. The observed stoichiometry provides strong support for an advanced model of glial–neuronal coordination, linking glucose consumption and glutamate neurotransmitter cycling, which was originally proposed from research on cellular suspensions (Pellerin and Magistretti, 1994), and has been shown to be the major pathway in vivo (Magistretti et al., 1999).

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Vcyc (µmol/g/min) 13 Figure 16.2. Relationship between Vcyc and CMRN C MRS experglc(ox) established from experimental results of iments at 7 T (Sibson et al., 1998) where rats were moderately anesthetized (with morphine and α-chloralose) to isoelectric conditions (high-dose pentobarbital). The best-ﬁt line is given by Equation (16.1) (in units of µmol/g/ min), which suggests that each mole of neurotransmitter glutamate cycling requires the oxidation of 1 mol of glucose. The awake, resting state has CMRN glc(ox) of approximately 0.8 µmol/g/ min, which means that under this condition ∼80 % of the resting energy consumption is dedicated to events associated with Vcyc . (Modiﬁed from Sibson et al., 1998).

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16.2. THE RESTLESS BRAIN The functional integrity of the brain depends on a collection of active cells which are continuously transferring and processing information. Our studies have established the relationship between neuroenergetics and neurotransmission most quantitatively in animal models and similar results ﬁt the available human data (Rothman et al., 1999, 2002). Our major ﬁndings can be summarized as follows: 1. Changes in energy consumed are coupled to the ﬂux of glutamate neurotransmission (Chapter 5) and neuronal ﬁring (Chapter 10) over a wide range of cortical activity (see Equation (16.2)). 2. In the awake condition (i.e., baseline activity), more than three-quarters of the brain energy is efﬁciently devoted to neuronal activity in the absence of external stimulation (Chapter 5). Cognitive or sensory stimulation can change this activity slightly. 3. A speciﬁc level of neuronal activity is required to perform a task. Increments detected by functional imaging do not measure the energy supporting the task but only the difference from baseline (Shulman et al., 1999: Hyder et al., 2002: Chapter 9). 4. The incremental functional imaging signal upon stimulation can localize regional changes in activity. However, the absence of an incremental signal does not mean the region does not participate in the function, rather that the activity does not differ from the baseline state. 5. Based upon these results we propose in this chapter that consciousness, deﬁned as the individual’s awareness of the world and of his subjective experience, tracks the degree of total brain activity. Most of the activity responsible for consciousness is discarded in a conventional functional imaging experiment.

16.3. IMPLICATIONS FOR FUNCTIONAL IMAGING These results allow new neurophysiological and neuronal interpretations of contemporary functional imaging experiments. Two possible interpretative pathways of functional imaging protocols are shown in a schematic presentation (Figure 16.3) taken from an earlier study (Shulman and Rothman, 1998). As indicated by the top line, the signal S, measured in a functional imaging experiment can be interpreted to provide insight into brain functioning M. The signals may derive from sensory experiments, or from more psychologically based protocols, but in either case they are interpreted in terms of a psychological model to elucidate brain function directly. The insights provided by this upper pathway are responsible for the popularity of functional imaging experiments. However, from our viewpoint the main explanatory pathway is diagrammed in the lower lines. In that approach, the fMRI or PET functional imaging experiments can be interpreted, usually in conjunction with ancillary data, to determine the neurophysiological parameters (NP) of brain energy consumption, most conveniently expressed as CMRO2 . Our results have shown that the values of CMRO2 determine neuronal activity, (N) expressed either as neurotransmitter cycling, Vcycle , (Chapter 5) or neuronal spiking frequencies (see Equation (2) of Smith et al., 2002 and Chapter 10). This explanatory pathway provides thermodynamic approaches to the questions of brain function M and reach different conclusion about the brain than those used in the psychological approach represented by S–M. The ease of measuring incremental activity by functional imaging has been consonant with the widespread view that increments localize the modular energy required for functional activity (Posner and Raichle, 1998). Our ﬁndings (Chapter 10) show that these increments arise from changes across the total neuronal populations whose sum changes the localized activity. The magnitude of stimulus-induced incremental activity in awake human experiments is much smaller than the basal activity. In anesthetized animals the size of incremental activities depend on depth of anesthesia and can be larger (Shulman et al., 1999) but

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Figure 16.3. Schematic relationship between the neuroimaging signal (S) and mental processes (M). In the usual experimental plan and interpretation, which is inﬂuenced by psychology and cognitive neuroscience, a direct relationship between S and M is assumed, as represented by the upper Psychology pathway (dotted line). While the deﬁnition of M is based on psychology, the experimentally derived S serves to localize and quantitate the brain activity identiﬁed with the process. The lower Neuroscience pathway, assumes that M has a molecular and cellular basis, which is broken into three steps leading to S. The signal, S, in fMRI or PET experiments, is primarily a measure of the neurophysiological parameters (NP) of CMRglc , CMRO2 , or CBF, as shown by the gray line. The relation between (NP) the neurophysiological measure of energy consumption and neuronal activity (N), which is shown by the dark line, has been clariﬁed by the 13 C MRS (Sibson et al., 1998) and electrophysiology (Smith et al., 2002) experiments. These recent ﬁndings allow measurements of S to be converted into measures of N, which places us squarely facing the unsolved ‘hard’ problem of neuroscience, i.e., what is the relationship between M and N (dashed line)? (Adapted from Shulman and Rothman, 1998).

are still generally smaller than the baseline. Considering that a speciﬁed task is supported by the magnitude of incremental activity neglects contributions of basal activity and is misleading for two reasons (Shulman and Rothman, 1998). First, for example, if CMRO2 /CMRO2 in a localized region for one cognitive task is 1 % (a typical value) and for another task it is 2 %, it would be misleading to conclude that twice the neuronal activity is required in the second task. Since approximately 75 % of CMRO2 is associated with neuronal activity in humans (Shen et al., 1999; Lebon et al., 2001), a more accurate conclusion would be an increase in neuronal activity from 76 % to 77 % of the total energy consumed. Second, differencing discards high regional activity which is required in response to a stimulus (Hyder et al., 2002). This required activity includes, in our interpretation, subjective contributions, which invalidate the staring assumptions. The subjective contributions including, for example, emotions, associations and experience, play the role in cognitive experiments that the ‘Moose’ fulﬁlls in business. The ‘Moose’ is the widely acknowledged big problem that is not discussed (McNeil, 2003). Some executives show leadership qualities by ‘putting the Moose on the table’ and discussing the big problem, but very few cognitive psychologists have analogously acknowledged these confounding contributions to cognitive tasks (Fodor, 2000). Consequences of the large baseline activity determined by 13 C MRS studies (Sibson et al., 1997, 1998) for interpreting neuroimaging data have been discussed previously (Shulman and Rothman, 1998). This interpretation provided a simple explanation of the negative difference signals, observed with fMRI and PET, which at that time had been considered an anomaly, (Raichle, 1998), from the assumption that baseline activity was negligible. On the basis of high baseline brain activity negative signals in a localized region were interpreted as a stimulated reduction in regional activity (Shulman and Rothman, 1998). More recently, the existence and the importance of signiﬁcant neuronal activity during nonstimulated conditions as a special property of certain brain regions (based on a meta-analysis of neuroimaging data) has been

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recognized by Gusnard and Raichle (2001). However, in contrast to the 13 C MRS measurements of a ubiquitous high resting brain activity, efﬁciently supporting neuronal activity, Gusnard and Raichle have proposed the existence of speciﬁc localized activities at rest in limited cortical regions. Their assignment follows from their ﬁnding that negative difference signals occur in certain regions to which they accordingly attribute high cognitive activities at rest. Their postulated ‘baseline’ activity also differs in kind from the widespread high activities determined from the 13 C experiments in that they claim a psychological basis (the difference observed between two psychological states) rather than a quantitative neuroenergetic basis for the existence and identiﬁcation of baseline activity (they have named the Moose). We now discuss how the high baseline activity provides an approach to brain function that goes beyond the Posner and Raichle (1998) interpretation of fMRI, to uncovering previously inaccessible brain activities.

16.4. NEURONAL BASIS OF BASELINE AND INCREMENTAL SIGNALS The 13 C MRS experiments (as well as ﬂuorodeoxyglucose (FDG) experiments by PET) can separately measure either baseline or incremental signals, which represent activities of a very large population of neurons. Electrophysiological brain studies have suggested that the collaboration amongst a large number of neurons, ﬁring at different frequencies, is crucial for encoding information in the brain (for a review see Scannell and Young, 1999). The idea that a certain number of neurons in a cortical region respond to a given stimulus (i.e., population voting) is not new. It was suggested long ago by E.D. Adrian who observed that touch causes discharges in several nerve ﬁbers, with relative intensities that map the stimulus onto the cortex (for a review Kandel et al., 1991). Information is more likely to be encoded in the brain by neuronal populations than by actions of individual neurons (for a review see Kristan and Shaw, 1997) because typically each cell responds to more than one stimulus variable (e.g., intensity, frequency, etc.), and an individual cell cannot distinguish amongst these different stimulus variables. Since the response proﬁles of neurons are different, and a single neuron does not participate in just one type of function, the ﬁring of a single cell by itself does not provide a meaningful basic code for information processing (for a review see Chapin and Nicolelis, 1999). The 13 C MRS measurements of high resting baseline activity are the sum of energy consuming connections measured in the voxel between neurons of different types (Chapter 10). In addition to local interneurons the synaptic activity in a voxel responds to connections with the rest of the brain (Braitenberg and Schuz, 1991). These remote neuronal connections, which support the delocalized aspects of brain activity, contribute to the total activity. Finally the somatosensory cortex has direct input from the sensory neurons via the thalamus – an input that is measured in the experiment. The resting unstimulated neuronal activity, which arises from local and distal neurons, is reduced by anesthesia both inside and outside of the somatosensory region. Upon stimulation of the anesthetized rat, the total neuronal activity inside the somatosensory voxels increased whereas total neuronal activity outside the stimulated region remained low (Ueki et al., 1988, 1992; Hyder et al., 1997). Thus the forepaw stimulation increased neuronal activity in the somatosensory cortex, but since the rest of the brain continues to show low activity its transmission to the somatosensory region remains low. This conforms to our observation that under strong stimulation the somatosensory ﬁring, seen in the neuronal histograms, is the same at our two levels of anesthesia so that it responds mainly to the sensory input (Smith et al., 2002; Shulman et al., 2002; 2003). In a nonanesthetized experiment the situation seems different. In the nonanesthetized awake brain at rest, the forepaw stimulation raises the total neuronal activity only slightly (Shulman et al., 1999; Hyder et al., 2002). Preliminary results, extending the α-chloralose experiments of Smith et al., using a lighter level of anesthesia, have shown departures from the histograms of populations with and without sensory stimulation (Hyder, Chapter 10) suggesting that the remainder of the brain is beginning to contribute signiﬁcantly. The interplay between contributions from localized and delocalized neuronal connections is measured by these

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experiments, with the promise that experiments varying the parameters of sensory input, anesthesia depth and the cortical level probed will provide additional insight to important questions of brain organization. The pattern of neuronal ﬁring, speciﬁc down to the level of individual neurons, is created, in these deeply anesthetized stimulated conditions, by the stimulus. The change in total energy upon a stimulus, detected as the difference signal, S, is a determinant of the signal usually reported in an fMRI experiment. The usual assumption of an fMRI experiment, i.e., that S reﬂects an involvement of that localized activity, is consistent with these neuronal results and is the basis of the usefulness of the differencing signals. Difference signals reﬂect an integrated, localized change in neuronal activity caused by the stimulus. However, the magnitude of the increment depends upon the relative activities in baseline and stimulated states, so that it does not measure brain activity needed to support the stimulus. Furthermore, neglect of the large baseline activity additionally invalidates the magnitude of the increment as a measure of brain activity. This kind of differencing, which is the basis of cognitive functional imaging experiments, has the form of a paradigmatic scientiﬁc experiment (Shulman, 1996). From a psychological or cognitive basis, a hypothesis is proposed that a task will change brain activity in a localized region and an experiment is performed to see if this is observed. In contrast, when considering the total or baseline brain activity such a formal structure of cause and effect is not available. The total level at rest, being identiﬁed as the baseline level – without any speciﬁc controlled sensory input – we consider to have no objective cause. There are no psychological or cognitive concepts that we would postulate to cause this activity, since there is no identiﬁed stimulation. We would not consider it scientiﬁcally valid, for example, to hypothesize that the brain, in this kind of unstimulated resting state, was engaged in or dedicated to a particular, identiﬁable activity, e.g., memory, love or computing. Without a causal claim, the baseline activity contrasts with the difference activity in a cognitive experiment, where an orchestrated event is postulated to cause the difference signals measured.

16.5. CONSCIOUSNESS AND BASELINE ACTIVITY In agreement with many efforts to review and deﬁne consciousness we have no exact deﬁnition to propose. Zeman has reviewed a wide range of the concepts of consciousness and found that two deﬁnitions dominate the usage. To be conscious in the ﬁrst condition is taken to be awake, aroused, alert or vigilant (Zeman, 2001). This aspect of consciousness describes how we communicate with our environment including others. The second concept emphasizes the ‘qualitative subjective dimension of experience’. We do not try to distinguish a priori between these two conceptualizations and for the moment identify both of these activities of mind with neuronal responses. Our question is how does the high neuronal activity, measured in the absence of speciﬁed external stimuli, relate to consciousness? In a start to answer this question we consider the quantitative relations between, and empirical deﬁnitions of, the degree of brain activities and the degree of consciousness used clinically by anesthesiologists. Our biophysical results measure the rate of energy consumption evaluated by CMRO2 , the rate of neuronal signalling measured by neuronal ﬁring, ν, and the rate of neurotransmitter release Vcycle . PET measurements have reported that glucose metabolism decreases with deepening anesthesia for several anesthetics (for a review see Heinke and Schwartzbauer, 2002). These reductions are generally global although small regional differences are found. Titrating human glucose consumption with isoﬂurane, halothane and propofol anesthetics ‘to the point of unresponsiveness’, the whole brain rates of glucose metabolism measured by PET were decreased by 40 %, 46 % and 55 % respectively (Heinke and Schwarzbauer 2002; Alkire, 1998; Alkire et al., 2000). These decreases in metabolic activity were ‘believed to reﬂect the reduced synaptic activity’, as judged by EEG variables. Our relationships (Equations (16.1) and (16.2)) now establish this connection with great certainty, since the available measurements of glucose metabolism are now quantitatively linked to neuronal ﬁring (Chapter 10). Along the pathway of deepening anesthesia, upon reaching about one half the normal brain ﬁring rates,

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the subjects reached ‘the point of unresponsiveness’, where they have, by these criteria, lost consciousness. This clinical deﬁnition includes both of Zeman’s concepts of consciousness as subjects were no longer aware nor did they report subjective experience. Therefore there is a quantitative correlation between both commonly accepted qualities used to describe consciousness and the magnitude of neuronal ﬁring. While these correlations may be further reﬁned by taking advantage of the spatial resolution of functional imaging methods (Alkire et al., 2000), we emphasize here the ﬁrst order correlation between the averaged level of brain activity and the degree of consciousness. This correlation could be expanded by associating intermediate levels of neuronal activity with varying degrees of consciousness. Decrease of the baseline activity with deepening anesthesia and the accompanying loss of consciousness establishes the importance of high baseline activity. It is connected, in ways that we are only now able to start exploring, with both aspects of consciousness. In the differencing methods of functional imaging the baseline activities are discarded, only the differences between two cognitive states being retained. In this respect the differencing imaging methodology parallels the assumptions of cognitive psychology, in which, as has been shown (Searle, 1992; Fodor, 2000), subjective aspects of consciousness are neglected.

16.6. NEUROPHYSIOLOGICAL STRUCTURES Our experimental results have measured two kinds of neuronal activity. One, arising from a speciﬁc sensory stimulation, causes a redeployment of neuronal ﬁring, which changes the total energy consumption of a localized brain region. This energy difference can be detected in a functional imaging experiment as an incremental change. The other kind of neuronal activity takes place in the rest of the brain, outside of the sensory cortex that responds to the stimulus. This delocalized outside activity is strongly reduced in the anesthetized state, and it is not increased during stimulation of the anesthetized animal. A relevant neuroscientiﬁc framework for relating localized and widespread activities has been offered and developed by Dehaene and colleagues (Dehaene et al., 1998; Dehaene, 2001). They assumed the participation of large neuronal populations in brain function from measurements of electrical activity in awake and asleep humans (Changeux, 1985). They have conceptually divided neuronal activity into a speciﬁc localized response to brain tasks (in our experiments this would be to forepaw stimulation) which interacts with a more delocalized response that modulates and controls the speciﬁc response. They deﬁne the delocalized activity as a global workspace that can ‘potentially interconnect multiple distributed and specialized brain areas in a coordinated, though variable manner, and whose intense mobilization might be associated with a subjective feeling of conscious effort.’ (Dehaene et al., 1998). They propose the global workspace to be active when, in addition to the speciﬁc modules, a stimulus interacts with other brain regions (Figure 16.4). The baseline activity measured from layer 4 in the absence of stimulation is identiﬁed as input from, and connections to, other neurons including from those in regions of the global workspace (Shepherd, 1994). The widespread decrease of baseline activity by anesthesia reﬂects a decrease in global activity, while the similar low baseline activity in the sensory cortex shows low input from the global workspace in the absence of peripheral stimulation. Outside the somatosensory cortex the brain remains at the low anesthetized level of activity during stimulation. Therefore input from the global workspace to the sensory cortex, should be reduced during stimulation in the anesthetized state compared to the non-anesthetized state. Consequently the high neuronal activity observed in the anesthetized somatosensory cortex during stimulation presumably is caused primarily by sensory input. This interpretation is supported by the almost identical histograms of neuronal ﬁring in the stimulated state at both levels of anesthesia (Smith et al., 2002; Shulman et al., 2003). By placing the electrodes in layer 4 of the somatosensory cortex the responses are weighted heavily towards the sensory input arriving via thalamo-cortical axons. The nearly identical histograms at two levels of rather deep anesthesia are in accord therefore, with expectations that the neuronal population is responding primarily to the sensory stimulation. The entire population of neurons selected

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Sensory cortex

Global workspace

Figure 16.4. Schematic conﬂuence of local and global ﬁnings in the sensory cortex.

responds to the stimulus in a ﬁxed way at these deep levels of anesthesia where there is essentially no input from the global workspace. Early results from lighter levels of anesthesia, halothane, do not give the same histogram of ﬁring (Hyder, personal communication). These results are consistent with a very speciﬁc neuronal deployment responding to sensory stimulation and suggest that the ﬁring pattern is a response to some integration of inputs from sensory and global ﬁring. The nature of this integration, and the assessment of the components, can be examined by varying the experimental parameters of (1) amplitude of stimulus, (2) global activity and (3) somatosensory cortical layer probed. These opportunities encourage us to expect progress in understanding some aspects of the interaction between the objective sensory task and the intrinsically subjective global interactions. In these assignments of total energetics and activities, the experiments described in this paper can provide quantitative measures to help separate contributions from local and global responses.

16.7. NEUROSCIENTIFIC MEASURES OF BASELINE ACTIVITIES Differencing methods in functional imaging are consistent with psychological views of brain in which undeﬁned baseline activities can be neglected. The differencing methods proving so useful in functional imaging, both fMRI and PET, have obtained results that are ﬁtted comfortably into psychologies that neglect baseline activity and have thereby strengthened their objectifying concepts of brain activity. However, the ranks of psychologists and brain scientists with more inclusive views of brain, have also been swelling recently. These researchers are of the opinion that cognition considers only a small fraction of brain activity. The high level of brain activity in the absence of any conscious stimulus shown by our results is consistent with those psychologies which include these kinds of activity. Gerald Edelman has written and experimented extensively in support of his views of the brain–mind relationship. In a brilliant, scholarly postscript he has criticized views that claim mind can be fully described by the representations of cognitive psychology. Framed by his view that cognitive psychologists have disregarded ‘a large body of evidence that undermines the view that the brain is a kind of computer’, he has delineated the nature of interactions between neurons, which he characterizes as re-entrant pathways. Without summarizing his broad studies we only note that the magnitude of baseline activities which we measure and attribute, in large part, to subjective experiencing would accommodate the continuous deep neuronal interactions that Edelman has proposed (Edelman, 1992).

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Damasio in a series of books (Damasio, 1999) has also concluded that cognition must be considered as only part of the far reaches of the brain that includes emotions and extends to interactions with the body that are inseparable from brain activities. It is also clear that criticisms claiming Freud’s work has been contradicted by modern brain research in cognitive neuroscience (Kitcher, 1995) are not supported by our ﬁndings. Rather, Freud’s work, based upon the existence of high levels of brain activity, operating dynamically with much of it unconscious, would be consistent with our high baseline activities in contrast to more recent theories where subjective activity is discounted. In addition to these claims for broader views of brain including, in some guise or other, high unstimulated brain activities, several neuroscientists have developed models of brain activity from observed baseline activities that are consistent with the high activities we have quantitatively measured. The need for substantial unfocussed neuronal activity for the service of even sensory responses was suggested by the brilliant experiment of Grinvald and colleagues (Tsodyks et al., 1999). Starting with the recognition that ‘cortical neurons are spontaneously active in the absence of external input even in primary sensory areas,’ they studied the correlations between single-unit recordings and real-time optical imaging. While interpreting their data, they concluded by suggesting that ‘in the absence of stimulation the cortical network wanders through various states represented by coherent ﬁring of different neuronal assemblies’ and that a stimulus pushes the network into a particular activated assembly. Analogously, the elegant temporal synchronization of neuronal responses on which Singer’s conclusion are based has led to the conclusion, ‘Of the many responses of VI those that become synchronized best will be particularly effective in inﬂuencing neurons in higher areas (Singer, 1994).’ This hypothesis recognizes the large neuronal activity in the absence of stimulation and includes that activity in proposals about brain function. In a similar vein also emphasizing the importance of neuronal oscillations, Llinas (2002) starts from the view, ‘that nervous system function may actually operate on its own, intrinsically, and that sensory input modulates rather than informs this intrinsic system’. The interaction between sensory cues and the high existing brain activity allows a larger view of brain function. In contrast to localized computer-like modules, Llinas has proposed that synchronized ﬁring of thalamo-cortical connections form the content of a highly active brain and play an important role in deﬁning the subjective self. Our 13 C NMR results, measuring high baseline activities can provide a basis for the assumptions made by these other brain studies, and offer a measure of their magnitudes.

16.8. NEED FOR PHILOSOPHY We have touched upon differences between the interpretation of functional imaging experiments in terms of cognitive psychology and the interpretations of the same experiments to explore the neuronal activities responsible for the signals. While these extremely different approaches are readily recognized as reﬂecting different perspectives, less dissimilar scientiﬁc positions also differ more than are commonly recognized. In neuroscience, different subﬁelds of science provide methods and approaches that require different starting assumptions and seek different types of understanding. In order to distinguish these diverse approaches it is necessary to examine their origins closely to see what their results mean, and upon what assumptions they depend. In contrast to many physicists who ﬁnd no need for philosophy in a science where epistemological methods and metaphysical assumptions are generally accepted, in neuroscience philosophical analysis is necessary in order to distinguish the differing basis of starting assumptions, methodology, and epistemological goals. Our separation of brain activities into a differencing activity and a baseline or total activity, where the former arises from a cognitive task and the latter from wide unspeciﬁed internal brain connections, ﬁnds a parallel in David Chalmers’ philosophical analysis of brain activity or consciousness. Chalmers has suggested that consciousness could proﬁtably be divided into ‘easy’ and ‘hard’ problems (Chalmers, 1996). Easy problems would be those that can be addressed objectively, such as the studies of sensory or cognitive

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tasks which are evaluated in functional imaging experiments by the difference signal. Hard problems that would help to explain subjective experience, he suggests are beyond our present ability to formulate. These subjective experiences, we suggest, are expressed in the totality of neuronal activity, whose connections, at present, cannot be formulated in causal terms. We propose that the present approach, which does not claim to explain subjectivity, still does offer an experimental entry for exploring these unknown areas of subjective brain activity, and for measuring their magnitude. Can philosophers not only help us to understand what scientists are doing but also provide useful advice? Chalmers has suggested that scientists might do well to look for an understanding of the hard problem by starting with cognitive studies of related qualities such as awareness. He says (Chalmers, 1996), ‘consciousness and cognition do not ﬂoat free of one another but cohere in an intimate way’. This direction has been followed by many respected cognitive scientists. Starting with difference signals, they have looked for interactions between the signals found from the effects in the visual response of attention being paid to nearby stimuli (Kastner et al., 1998) while others have taken correlations between difference signals in different brain regions (Friston, 2002). These attempts to extend the meaningfulness of fMRI beyond the objective assumptions of cognitive psychology are in accord with Chalmers’ suggestion that cognitive conceptualizations can provide a good starting point for studying related subjectivities. But we have chosen a different direction to study the ‘hard’ problem, a direction which starts with hard, reliable assumptions based on physical knowledge. We have measured the relationship between energy consumption and the activity of local and distributed neurons. Our starting points have been quantitative and unambiguous and the questions they ask have been answered with ‘hard’ data. From the secure position these results have generated, we have proposed to advance slowly, but sure-footedly, towards the exploration of previously inaccessible neuronal functions. Thomas Nagel (1986) has focussed philosophical studies upon ‘how to combine the perspective of a single person with an objective view of that same world.’ Because of the pervasiveness of the two different views any reconciliation or accommodation would form the core of a worldview. He disavows the aim of uniﬁcation being unwilling to settle for its simpliﬁcations, and he prefers the perplexities generated by a frontal juxtaposition of subjective and objective. If developed separately into a worldview he discusses how each would suffer from incompleteness. He claims, for example, that existing objective studies must leave the subjective human perspective behind. Physics, he continues, where we have achieved ‘our greatest detachment from a speciﬁcally human perspective’ for that reason ‘is bound to leave undescribed the irreducible subjective character of conscious mental processes’. This limitation has been countered by many and most directly by John Searle (1982) who argued that just because the mind is intrinsically or ontologically subjective does not mean that objective study is incapable of understanding its subjectivity. Difﬁcult and complicated but not impossible. Nagel brings us close to the present scientiﬁc problem when he suggests that the modern acceptance of physicalist reduction has not been the answer since ‘a less impoverished and reductive idea of objectivity has not been available’ to provide the understanding he seeks. This brings us to the present study. We do not, of course, ﬁnd physics a burden to understanding but quite the opposite, it is the very basis we are celebrating. In agreement with Searle we ﬁnd the early steps in the road the most challenging and feel that, as described above, we are on a path towards some objective measures of subjectivity. How far it will go we have no idea. But if we wish to compare the present state of neuroscientists with a similar moment in history we could do worse than to reﬂect upon the Scientiﬁc Revolution of the 17th century. To provide a perspective on the varieties of options available for modern neuroscientiﬁc research we select a few features of the innovations introduced by Renee Descartes in the early years of modern physical science. Those issues focussed the state of physical science during the Scientiﬁc Revolution of the 17th century. With its exciting hopes for future understanding, and with

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its nascent methodologies Descartes’s time has many similarities with the conditions prevailing in today’s neuroscience. Changing methods went hand in hand with philosophical re-evaluations to give birth to physical science. Descartes, whose life spanned the ﬁrst half of the century played a pivotal role in the transition from the older ways of scholasticism, where novelty was expressly forbidden, to the empirically testable hypotheses of modern science. In 1637 Descartes published his Discourse on the Method of Properly Conducting One’s Reason and of Seeking the Truth in the Sciences (Descartes, 1637), which started with his disappointment, in ﬁnding after much study, ‘that there was no body of knowledge in the world such as I had been led previously to believe existed’. Philosophy was useless since it contained nothing that was not still in dispute. ‘As for the other sciences, in so far as they borrow their principles from philosophy, I consider that nothing solid could have been built on such shifting foundations’. He thereby abandoned two millennia of Aristotelian science, incorporated into theology as Scholasticism, and risked being condemned for heresy, as Galileo had suffered 4 years earlier. In this skeptical state, Descartes conﬁdently resolved to ignore available knowledge completely and to start fresh. He proposed rules for arriving at knowledge. The method started by assuming that which is ‘clear and distinct’ and the questions raised by this individualistic vision are then reduced, by Descartes’ method, to their components so as to understand the original whole. Attention is more usually paid to Descartes’ method of analysis, which was, in fact his subject, and particularly to its concluding dualism, which bears directly upon neuroscience. However, we suggest that those particular consequences of Descartes’ method are of minor interest for the modern neuroscientist, who generally uses both Descartes’ methods and assumes that physical studies of brain will help to explain features of mind. Instead we wish to emphasize the revolutionary nature of his starting assumptions, which opened the way for modern physical science, and provide the kind of fresh start which, we suggest, we need in neuroscience today. In rejecting the existing scholastic view of nature, Descartes introduced a scientiﬁc worldview that became the method of physical science. [The rapid replacement of ‘clear and distinct’ by modern scientiﬁc hypothesis, susceptible to empirical disproof has been traced by many (see Clarke, 1992)]. Neuroscience has been created in the expectation that scientiﬁc methods can help to explain the qualities of mind, thereby extending the successes of physical science. We are not at a loss for the methods of analysis and experimentation, but in seeking scientiﬁc approaches we are struggling with conﬂicting starting assumptions. As summarized above, while studies of the brain–mind problem start from the objectivity of physical science, they must include subjectivity, which has been the purview of literature, psychology and other characteristically human concerns. Existing physical science does not provide a worldview that includes these opposites. We have not found the answer, but we feel that by quantitatively measuring the extent of brain activity that supports subjectivity, we are measuring the existence and magnitude of a physical parameter that is devoted to the complexities of subjective experience whose ﬁne structure cannot be presently separated. By considering that the brain is subject to the physical laws concerning energy and work, our assumptions are that the brain–mind is not different from any other material and that subjectivity is an intrinsic property of brain that cannot be neglected but whose magnitude can be explored objectively. We hope that an empirical research program based upon this intrinsic view of matter will make progress, and that, as empirical studies have expanded our physical understanding, ﬁrmly based physical studies of the organic matter that constitutes the body–brain–mind will continue to make novel understanding.

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17 The Role of the NMR Baseline Signal in the Study of Consciousness: the Restless Brain Robert G. Shulman Yale University School of Medicine, MR Center, P.O. Box 208043, New Haven, CT 06520-8043, USA

The research directions summarized in this book have converged to create an understanding of the brain’s baseline activity. Baseline is the awake activity that goes on in the absence of objective stimulation. When we require a subject to do a task involving memory or perception or understanding, any change in activity measured is relative to the activity that goes on in the brain in the absence of such stimulation. Two scientiﬁc directions, MRS and the calibrated fMRI data accompanied by spiking activity, provide a scientiﬁc understanding of the baseline which has consequences for activity of brain and mind. The results describe a high amount of brain activity in the absence of external stimulation. As shown in Chapters 5 and 10, the baseline activity is decreased with depth of anesthesia as the amount of energy consumed and the neuronal ﬁring rates decrease. From the MRS experiments this activity accounts for the majority of the energy consumption of the brain. The brain in this sense is very efﬁcient, making good use of its energy consumption for its function, namely to ﬁre neurons and to handle information. We have named the brain exhibiting this baseline activity the restless brain. The second important attribute of baseline activity, shown in Chapter 5 and extended in Chapter 10, is that high baseline activity is needed for the performance of a task. During stimulation of the animal from two different levels of anesthesia the energy consumed and the neuronal ﬁring rate reached the same total levels during stimulation regardless of the depth of anesthesia. Hence the total activity, of the order of the resting baseline activity, is needed for the animal to respond to a sensory stimulus. Not only is the baseline activity high but a similar level of high activity is needed for the animal to respond to a stimulation. Psychological concepts, particularly from cognitive neuroscience, have played a major role in the formulation and interpretation of functional imaging experiments. The major operative concept for image interpretation is that the functional localization of the brain can be mapped based upon regional changes in brain activity. The many images obtained have been considered afﬁrmation of the valuable nature of the starting hypothesis. However, as indicated in Chapter 10, a different approach to functional imaging Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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experiments is suggested by our present understanding of baseline activity. First the baseline contains valuable information which is ignored or discarded in a functional imaging differencing experiment. Second the baseline activity which is discarded is of the same nature – namely neuronal – as all the increments which are retained. Third the difference signal in a functional imaging experiment represents not the energy needed for the brain to perform a task but the difference in energy and activity between the conditions of that task and the baseline. The magnitude of this difference is not the magnitude of the brain activity needed to perform the task. Finally the localization of energy differences in a BOLD signal does represent a difference in brain activity between the baseline and stimulated conditions. Therefore, as so many functional imaging experiments have found, localization of a change is indicative of the participation of that brain region in the task. However, the absence of a change does not mean the region is not participating in the function. Differences of incremental signals in different regions of the brain do not measure their relative degree of participation in the task at hand. A larger incremental signal does not indicate a larger degree of activity being required for the task when the baselines may differ. In experiments starting from the same baseline magnitude differences do have meaning, but the role of total regional energy cannot be neglected. These ﬁndings logically need to lead to a re-evaluation of functional imaging experiments. Two chapters in the book have surveyed recent functional imaging experiments in the context of the other chapters of this book. In Chapter 14, Meltzer and Constable have surveyed the literature on declarative memory systems. Meltzer and Constable provide an updated search of experimental and conceptual studies of long-term declarative (LTD) memory – focused on the role of the hippocampus as found by functional imaging. The early fMRI studies failed to see incremental signals in that regional although the necessity of hippocampal function for LTD had been established by lesion studies. The authors acknowledge that a high baseline activity which did not increase signiﬁcantly would explain these results, an explanation quite consistent with the proposals in Chapter 16. Subsequent enrichments of the episodic declarative memory tasks in several ways such as by novelty and by the success of encoding did show incremental fMRI signals. A search to ﬁnd a unifying concept to conceptualize the incidental but often necessary additional task components was not successful in cognitive terms. Another example of where the standard functional imaging paradigm has led to paradoxical results, in which baseline activity is neglected, has been the reports of negative fMRI (and PET) difference signals during tasks. Negative signals were considered paradoxical because the activity during a task was implicitly assumed to be many fold higher than baseline. Our ﬁnding of a high baseline activity provided the obvious explanation of this phenomenon that had puzzled many. Gusnard and Raichle have recently explained the high baseline activity leading to negative signals using standard cognitive psychological concepts. Their explanation of the baseline activity is that it is caused by speciﬁc mental functions, related to awareness. In this way they have assumed an objective understanding both of the baseline condition and of the stimulated condition. On the other hand, we have offered a quite different explanation of the negative signals and of the high baseline activity. When a subject, in the magnet with eyes closed and the ears plugged, has no input from the experimentalist she is in a state that can only be described in subjective terms. We do not know by any objective criteria what that subject is thinking at that time. We prefer to lump all baseline activity together as not being objectively knowable for these reasons. In Chapter 15 Bruce Wexler has taken a broad view of functional imaging not only with respect to the recommendations of this book but also from the viewpoint of a practitioner who has reservations about some of the concepts being followed. His view is that there are limitations of the methods, which come not speciﬁcally from the neglect of baseline, nor from the use of differencing, but which come from the very nature of the objective knowledge being sought. To what extent, he asks, is the concept of localized brain activities really experimentally valid? He readily accepts the prevailing view that there are necessary interactions between regions that form a thought or a response, but he enquires about the experimental

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nature of the localization. Is the idea of localized function actually driving data interpretation, and possibly leading to unsubstantiated claims. Furthermore he goes on to examine the meaningfulness of brain responses in view of the changing brain activities with time, e.g. the time of the experiment, the conditions of the subject at the time, the changes within the subject in short times, with familiarity of the protocols, and the contents. Furthermore subjects also change with time, with growth, with aging, with development. All these considerations lead to an acknowledgement of the complexity of brain function which is consistent with the argument, made in Chapter 15 that we should be less conﬁdent of our psychological starting points for brain studies. The high baseline activity in the absence of stimulation and its requirements during stimulation have great consequences for an understanding of the brain–mind relationship. The relation of brain and mind is the traditional subject of psychology. It is our contention that these neuroscientiﬁc measurements of baseline provide constraints for what a proper approach to psychology should be. In looking for explanations of the higher activities of brain–mind from scientiﬁc results the most successful ventures in the past have shown how particular activities, considered to be those of mind, can be explained scientiﬁcally. The results of neurotransmitter receptor agonists, the consequences of hormonal release and the suggested explanations of long-term potentiation are but a few of the many scientiﬁc results that have been shown to have a bearing on the more complex activities of mind. However, this aspect of our approach is more metaphysical and directed to the level of what a valid explanation would be rather than to speciﬁc features of brain activity. We have proposed that scientiﬁc results require a proper framework for the study of mind, and that this framework should acknowledge the importance of subjectivity at the outset of the experiments, such subjectivity being supported by the high, unassigned brain activity measured at baseline. A clear separation between the subjective and object of aspects of brain–mind was made by Francis Crick in his book The Astonishing Hypothesis, which reinvigorated the scientiﬁc investigation of consciousness. Crick proposed that we should study the brain objectively and postpone any considerations of the subjective, which he lumped as Qualia. This reasonable traditional scientiﬁc approach, of selecting parameters, has been followed by many psychologies. However, it is our position that ignoring subjective aspects of brain–mind activity is ﬁrst of all misleading as a beginning and second, in light of our present scientiﬁc results, is no longer necessary. It would take us too far aﬁeld at this point to categorize different psychological approaches by their attitude towards objective/subjective. We have found that, as a working hypothesis, the concept of a global workspace to include subjective experiencing is extremely useful. Chapter 10 indicates how future experiments can continue to explore particular aspects of subjectivity in a quantitative neuroscientiﬁc fashion. At present these different assumptions about the nature of baseline activity are a matter of opinion. It is perfectly valid, as represented by Crick’s stand and by Gusnard and Raichle’s approach, to expect that unexplained areas of brain will be explained successfully by objective hypotheses that can be proposed now and be tested. However, the ability to explore and to measure the contents of resting baseline activity without postulates as to the speciﬁc meaning of their contents is a promising approach not previously available. NMR makes it possible to begin exploring these unknown areas without postulating in advance what they may be. Rather we can now measure their magnitudes and the pathways they follow, e.g., cortical thalamic pathway versus cortical, and see if by these explorations of the unknown areas of brain activity we can come to unexpected exploratory hypothesis. In this way hypotheses will be guided more by the neuroscientiﬁc ﬁndings than by the psychological concept of how the brain works. Zeman in a recent review concluded that consciousness is generally used in two ways. In one it is used as synonymous with awareness. A person is aware of bright lights, of noises and these enter into one’s performance of other activities. This is an objective aspect of awareness and an objective aspect of consciousness and the word is often used in this meaning. The second meaning of consciousness is

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of subjective experiencing. This would include for example the inputs that the body is experiencing at any time but where the response of the body is below the level of conscious awareness. This is, in these respects, the unconscious aspect of consciousness. We propose that the total baseline activity supports both these aspects of consciousness. This is illustrated in Chapter 10 where Hyder and Blumenfeld present a neuronal response in the somatosensory cortex to a forepaw stimulation in the rat. The response at deep anesthesia, where only the sensory input is acknowledged by the neuronal ﬁring pattern, contrasts with the response at lighter anesthesia where additional input from the global workspace is seen. In these experiments, as described in Chapter 10, many empirical parameters can be varied in order to explore the interplay of conscious sensory input and the more global subjective response of the animal. In human experiments, the results of anesthesia show that at the point of nonresponsiveness, corresponding to the loss of consciousness, the human brain energy consumption has decreased by about 50 %. In the rat, of course, we do not have the subjective accounting that the anesthesiologist relies upon for the description of consciousness. However, anesthesia is used in the rat for the same reason that it is used in humans, namely to protect the animals from being conscious of pain. The dependence of subjective response upon the level of baseline activity allows the neuroscientiﬁc results to be extended into psychological considerations. These approaches provide an experimental opportunity to study the over-reaching interactions of brain and mind.

Index 3D imaging, CMRO2 139–141, 143 acetate 79, 87 action potentials biochemical mechanisms 17, 174, 175 cortical metabolic rate 112, 113 energy metabolism relationship 17–19, 27–28 glucose utilization relationship 16–17 grey matter propagation cost 115–116 spiking frequency 179–184, 186–187 activation maps 189 adenosine triphosphate (ATP) energy consumption distribution between signalling mechanisms 116–117 glutaminergic synapse metabolic cost 114 ion pumps 17, 19 phosphorus NMR measurement 37 total resting consumption 113 age effects on brain activation 288 alcoholism 246–247 Alzheimer’s disease 128 ammonia 83–84, 88 amnesia 263, 270 anaplerosis 79, 82, 83 anesthesia brain activity effects 302 CMRO2 and spiking frequency 179–184 glucose utilization effects 15 neurotransmission effects 298 stimulation response effects 179–184, 284, 301–302 astrocytes cerebral energy metabolism 78–81 functional activity–energy metabolism relationship 24, 28 glutamate effects on glucose utilization 25–27 glutamate–glutamine cycle 55, 81–93 astroglia (macroglia) 217–227 ATP see adenosine triphosphate attention shifting 289–291

autoradiography 13–14, 18 awareness/consciousness relationship 313 baseline neuronal activity consciousness 311–314 functional imaging 174, 188, 191–192 importance 304–305, 312 stimulus induced incremental activity comparison 299–301 blood oxygenation level dependent (BOLD) contrast CMRO2 and cerebral blood ﬂow 127–128 event-related fMRI 164 fMRI biophysics 151–153 fMRI calibration 153–156 MRI neuroimaging 149–151, 260–261 physiological basis 122, 156–160 brain cell types 54 brain damage 263, 270, 284–285, 291 brain function baseline activity 301–307, 311–314 changing cognition relationship 285–291 incremental signals 301–302 localized/ﬁxed models 280–285 oxidative metabolism role 126–128 subtraction study method 280–284 brainwork 4–5, 185, 186–7, 190 2-[14 C] deoxyglucose 13–14, 18 1-[13 C] glucose NMR compartment models 54–57, 68–69 data modeling 54–70 in vivo techniques 37–39 metabolic pools 57–60 model creation 60–62 model data ﬁtting 62–66 MR information content 66–69

Brain Energetics and Neuronal Activity. Edited by R. G. Shulman and D. L. Rothman  2004 John Wiley & Sons, Ltd ISBN: 0-470-84720-4

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C MRS cerebral energy metabolism 76–81 CMRO2 measurement 155–156 glucose oxidation 297 glutamate–glutamine cycle 83–89 intravenous catheters 249–250 neurotransmission energy requirements 89–93 neurotransmitter systems 297–298 psychiatric disorders 247–250 13 C NMR 37–39, 49–50, 67, 100–107, 129 Ca2+ sequestration 209–210 calibrated fMRI see functional magnetic resonance imaging CBF see cerebral blood ﬂow cell bodies 20–21 cell types 54 cerebral blood ﬂow (CBF) early in vivo metabolism studies xi, 12–13 imaging cerebral metabolic rate of oxygen consumption 127, 137–138, 139–141 imaging using magnetically labeled arterial blood water 155 positron emission tomography studies 162–164 relationship to CMRO2 148–149, 150, 159, 165, 176 cerebral blood volume (CBV) 128, 154–155 cerebral energy metabolism 75–93, 118–120 cerebral metabolic rate of glucose consumption (CMRglc ) 13–17, 148, 162–163 cerebral metabolic rate of oxygen consumption (CMRO2 ) 13 C NMR approach 129 calibrated fMRI derived changes 147–165 cerebral blood ﬂow relationship 176 change measurement 179–181 early in vivo metabolism studies xi, 12–13 imaging using 17 O NMR approach at ultrahigh ﬁeld 125–143 neuronal activity relationship 173–192, 260–262 positron emission tomography studies 128–129, 162–164 relationship to cerebral blood ﬂow 148–149, 150, 159, 165 relationship to CMRglc 148 spiking frequency relationship 181–184 chemical shift data 35, 36, 39 CHEmical Shift Selective (CHESS) 43 childhood epilepsy 204, 209, 227 children 204, 209, 227, 288 chloralose anesthesia 179–184 choline 198, 199, 200 clinical MRI applications 7–8

Index CMRglc see cerebral metabolic rate of glucose consumption CMRO2 see cerebral metabolic rate of oxygen consumption cognitive neuroscience 189–190 cognitive processes brain structure/function relationship 280–292 deep versus shallow tasks 265–266 fMRI interpretation 262 long term memory 259–277 cognitive psychology, criticism 304–305 cognitive subtraction approach 280–284 coils, 17 O RF 132, 138–139, 142 combination pools 60 condition I see light anesthesia condition II see deep anesthesia consciousness baseline activity 311–314 brain activity 299, 302–303 concept 302, 313–314 unconscious aspects 305, 314 consumption of energy 111–122, 283 correlation NMR spectroscopy see two-dimensional NMR spectroscopy cortical depression 17–18 cortical excitability 228–230, 243–244 creatine 198, 199, 200–202 creatine kinase (CK) reaction 37 data deﬂection 67 data ﬁtting 13 C NMR modeling 62–69 least-squares 62–63 numerical algorithms 63–64 scatter effects 64–66 declarative memory 262–264 deep anesthesia 179–184 deoxyglucose tracer molecule 5 deoxyhemoglobin 149, 151, 152 depolarization (Na+ inﬂux) 174 depressive disorder 245–246, 247–248 depth-of-processing effects 265–266, 267 Descartes, Ren´e 306–307 detoxiﬁcation, ammonia 83–84 differencing methods see also subtraction method baseline neuronal activity 176, 189–190 drawbacks 311–312 functional image interpretation 304 negative signals 312 stimulation versus baseline 302

Index disease 126–128, 291 see also brain damage; childhood epilepsy; epilepsy; psychiatric disorders electrodes 177, 191 electroencephalography (EEG) 177 encoding in long term memory 264–269 endocytosis 114 energy consumption 111–122, 283 energy metabolism 76–81 epilepsy astroglial metabolism 215–237 GABA role 227–232 glutamate metabolism 217–227 glutamate–glutamine cycle 219–220, 222–225 glutamine levels 225–226 healing process 204, 208–209 ketogenic diet 209 magnetic resonance spectroscopy 197–213, 215–237 astroglial metabolism 217–227 GABA role 227–232 1 H techniques 198–204 N-acetyl aspartate/creatine levels 198, 199–204, 208 31 P techniques 198–204 tissue heterogeneity 199, 201–202, 205–208 neuronal injury 202–204 seizure foci localization 198–199 seizure mechanisms 209–210, 232–233 episodic memory 264–265, 271–272 event-related fMRI 164 exocytosis 114 familiarity, memory studies 271–272 2-[18 F]deoxy-D-glucose (14 FDG) 14–15 Fick’s equation 134–135 ﬁeld potentials 174 ﬁring rate of grey matter 116, 121 ﬁxed brain structure/function relationship 280 ﬂuoroacetate 92 fMRI see functional magnetic resonance imaging forepaw stimulation 179, 181, 185, 189 Fourier transform NMR 32 fuel sources, monocarboxylic acids 54 functional magnetic resonance imaging (fMRI) biophysics 151–153 BOLD signals 260–261 calibration methods 153–156 CMRO2 change measurement 147–165, 180 development x-xii, 4

317 event-related 164, 266 human 162–164 interpretation 187–189, 299–301 long term memory 259–277 medial temporal lobe subdivisions 271–272 memory encoding 264–265 mind–brain studies 279–293, 299–301 neural basis 161–162 neural energy consumption 112, 121–122 neuroimaging 149–151 neurotransmission energy requirements 89 physiological basis 156–160 GABA (gamma-aminobutyric acid) catabolism 101, 102–103 cortical excitability 228–230 epilepsy role 216–217, 227–232 in vivo NMR studies 102, 106 neuronal localization 100–101 regulation 227–228 synthesis 102–104, 105–106 GABA shunt 102–103 GABA transporters (GATs) 104 GABA–glutamine cycle 13 C MRS 247–250 GABA neurotransmitter repletion 106 glial–neuronal interactions 240 glutamate/glutamine cycling 100, 101, 106 1 H MRS 241–247, 249 psychiatric disorders 243–248 GABAergic neurons 78–79, 81 GABAergic tone 243–244 GAD see glutamate decarboxylase gamma-aminobutyric acid see GABA GATs see GABA transporters GLAST (glutamate transporter) 92 glial cells see also astrocytes; astroglia function 54, 55 glial-speciﬁc enzymes 78 neuronal interactions 240–250 proliferation in epilepsy 224–225, 226 resting potential 113 tumors 219 global workspace 189, 303–304 glucose see also 1-[13 C] glucose; 2-[13 C] glucose; 2-[14 C]deoxyglucose; 2-[18 F]deoxy-D-glucose deoxyglucose tracer molecule 5 isotopic precursors 76, 78, 85–87 neurotransmission energy requirements 89, 90

318 glucose metabolism 13 C MRS measurements 76–78, 79 13 C NMR modeling 53–72 astrocytic oxidation 79 importance in brain metabolism 12–13 oxidative 126–128 psychiatric disorders 241 glucose utilization action potential relationship 16–17, 27 glutamate effects in astrocytes 25–27 local measurement methods 13–17 neuronal inhibition 23 glutamate see also glutamate–glutamine cycle clearance rates, epilepsy 218–219 effects on glucose utilization in astrocytes 25–27 epilepsy mechanisms 216–227 extracellular levels in epilepsy 209–210, 217–227 GABA cycle in epilepsy 230–232 intracellular levels in epilepsy 221–222 neurometabolic coupling 91–92 recycling 113–114 release, epilepsy 218–219, 220 glutamate decarboxylase (GAD) 102, 103–104, 106 glutamate pools 78 glutamate transporter (GLAST) 92 glutamate–glutamine cycle 13 C MRS 247–250 epilepsy 219–220, 222–225 GABA/glutamine cycling 100, 101, 106 glial–neuronal interactions 240 1 H MRS 241–247, 249 modeling 54–57 MRS measurements 81–93 neurone–astrocyte interaction 297–298 one-compartment model 56 psychiatric disorders 243–248 quantitation 297–298 two-compartment model 55, 68–69 glutamatergic synapses 113–115 glutamine see also glutamate–glutamine cycle GABA cycle in epilepsy 230–232 GABA synthesis 106 intracellular levels in epilepsy 225–226 neurotransmission 78, 89–91 transporters 104–105 grey matter 1 H MRS measurements 199–202 action potential propagation cost 115–116 cortical layers 178 energy consumption 112, 120–121

Index mean ﬁring rate 116 metabolic rate 92 1 H MRS studies see proton MRS hemoglobin 128, 134 heteronuclear spectral editing 49–50 hippocampal sclerosis see epilepsy hippocampus 265, 266, 270–272, 273 homonuclear spectral editing 46–49 human animal comparison techniques x hunger 289 Huntington’s disease 128 hyperammonemia 79, 83–84 hyperpolarization (K+ efﬂux) 174 hypothalamo-neurohypophysial pathway 20, 22

implicit memory 262–264 in vivo techniques 2-[14 C]deoxyglucose 13–14, 18 cerebral blood sampling 12–13 development x-xii 2-[18 F]deoxy-D-glucose 14–15 local glucose metabolism 13–17 NMR suitability 32, 33 incremental signals 259–277, 299–301 information processing 179 information representation 120–121 inhibition of neurons 23 inorganic phosphate 204–205 interactive processes 283 intracellular pH 37 intravenous catheters 249–250 ionic gradients 17–19, 27 isotope balance equations 60–61 isotopic labeling 12–15, 18, 53–72 isotopic precursors 76, 78, 79, 85–87 isotopomer analysis 61–62 J scalar coupling constant 45–49 K+ efﬂux 174 K+ extracellular concentration 17, 19, 24–25 α-ketoglutarate (α-KG) 84, 85, 86, 87, 102 Krebs cycle see tricarboxylic acid cycle lamotrigine 162 language 280–281 lateralization 266, 288, 291

Index LCmodel signal quantiﬁcation algorithm 43–45 learning 286–288 least-squares ﬁtting 62–63 ‘levels of processing’ (Craik) 265–266, 267 LFP see local ﬁeld potentials light anesthesia 179–184 local cerebral energy metabolism 13–17 local ﬁeld potentials (LFP) 177, 184 localization 280–285, 301, 313 long term memory (LTM) anatomical dissociations 262–264 depth-of-processing relationship 266, 267 encoding and retrieval 264–269 incremental signals 259–277 medial temporal lobe subdivisions 269–272 macroglia see astroglia magnetic ﬁeld homogeneity 40–42 magnetic resonance imaging (MRI) 4, 32, 149–151, 198–199 see also functional magnetic resonance imaging magnetic resonance spectroscopy (MRS) see also 13 C MRS; 15 N MRS; proton MRS cerebral energetics and neurotransmitter ﬂuxes 75–93 data modeling 54 development 32 epilepsy 215–237 epileptogenic tissue 198–213 1 H techniques 198–204 interpretation 202–204, 208–209 31 P techniques 204–209 sensitivity/selectivity 199–202, 205–208 importance xi-xii, 4 magnetoencephalography (MEG) 177 maintenance function (energy consumption) 120 mass balance equations 60–61 mean ﬁring rate of grey matter 116, 121 medial temporal lobe (MTL) declarative memory role 263, 264–265 incidental versus deliberate memorizing 268 long term memory 260 memory encoding 264–268 memory retrieval 268–269 subdivisions 269–272 MEG see magnetoencephalography memory see also long term memory cognitive processes 259–277 declarative 262–264 encoding 264–269

319 episodic 264–265, 271–272 types 262–263, 264 mental event representation 120–122 metabolism see also glucose metabolism 13 C MRS measurements 76–81 glutamate release regulation of energy 91–92 importance ix-xii rates modeling 59, 69–70 metabolite pools 13 C NMR modeling 57–60 brain cell types 54 NMR chemical shift data 35, 36, 39 NMR signal quantiﬁcation techniques 43–45 mind–brain relationship 8–9, 279–293, 295–309 mitochondria distribution and neural energy consumption 118, 119 dysfunction in epilepsy 204, 209–210, 232 metabolic H17 2 O 135 modeling analysis 13 C NMR studies 53–72 constraints and simpliﬁcation 61 data deﬂection 67 data ﬁtting 62–69 isotopomer analysis 61–62 mass and isotope balance equations 60–61 metabolite pools 57–60 relevance ratio 67–68 sensitivity 67 target data 60 monocarboxylic acids 54 mood disorders 245–246, 247–248 MRI see magnetic resonance imaging MRS see magnetic resonance spectroscopy MUA see multi-unit activity recording multi-compartmental models of metabolism 54, 55, 68–69 multi-unit activity (MUA) recording 177, 184 15

N MRS 88 N-acetyl aspartate (NAA) 1 H MRS measurements 198–204 13 C MRS psychiatric disorder studies 247–249 epileptogenic tissue 198–199, 200 1 H MRS psychiatric disorder studies 241–243 Na+ inﬂux, depolarization 174 Na+ intracellular concentration 17, 19, 24–25 NAA see N-acetyl aspartate (NAA) negative signals, high baseline relationship 312 neocortex anatomy 178–179 neural circuits 112, 122

320 neural energy consumption 111–122 neuroimaging see also functional magnetic resonance imaging interpretability of signals 175–177 MRI 4, 32, 149–151, 247–248 physiology 148–149 neurons activity brain structure/function studies 283–284 energy requirement 283 inhibition 23 injury/N-acetyl aspartate level relationship 202–204 loss in epilepsy 224–225, 226 oxidative metabolism in epilepsy 210 resting potential 113 types 54 neuropil 20–21, 23, 24, 27–28 neuroscience approach 295–297 neurotransmitters 13 C MRS ﬂux measurements 81–89 energetic requirements 89–93 functional activity–energy metabolism relationship 19, 25–27, 28 glucose metabolism relationship 298 glutamate–glutamine ﬂux studies 6–7 psychiatric disorders 239–251 recycling 113–114 neurovascular coupling 148–149 nitrous oxide method 12–13 NMR see nuclear magnetic resonance novelty 265, 267, 268 nuclear magnetic resonance (NMR) see also 1-[13 C] glucose NMR 13 C techniques 37–39 GABA neurotransmitter pathways 100–107 heteronuclear spectral editing 49–50 homonuclear spectral editing 46–49 importance ix-xii, 3–4 in vivo techniques 33–50 phosphorus techniques 34–37 proton techniques 33–34, 35 pulsed 32 scalar coupling 45–46, 47 shimming techniques 40–42 signal quantiﬁcation techniques 43–45 spatial localization techniques 39–40 spectral editing techniques 45–50 technique development 31–33 two-dimensional 45 water suppression techniques 42–43 numerical ﬁtting algorithms 63–64

Index 17 O NMR 129–143 object recognition 283 older adults 288 one-dimensional NMR spectroscopy 45 osmotic stimulation 20, 22 ouabain 92 oxidative metabolism 126–128 oxygen 12, 129–143 see also cerebral metabolic rate for oxygen

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P MRS 204–209 paramagnetism of deoxyhemoglobin 149, 151, 152 Parkinson’s disease 128 PCr/Pi ratio 204–205 ‘perturb and detect’ philosophy 186 PET see positron emission tomography pH effects 37 philosophy 305–307 phosphate, inorganic 204–205 phosphorus MRS 204–209 phosphorus NMR 34–37 physiological states 289 POCE (Proton-Observed, Carbon-Edited) NMR spectroscopy 49, 155–156 polyamines 104 positron emission tomography (PET) CMRO2 , (CMRglc ) and CBF changes in awake human brain 162–164 CMRO2 in vivo determination 128–129, 134 development x, 4 2-[18 F]deoxy-D-glucose method 15 functional imaging 299–301 medial temporal lobe memory role 264–265 post-synaptic energy consumption 114, 115, 117 potassium ions see K+ prefrontal cortex 264 proton (1 H) MRS studies epileptogenic tissue 198–199 glutamate role in epilepsy 220–221 glutamate–glutamine-GABA cycles 243–247, 249 in vivo techniques 33–34, 35 N-acetyl aspartate 241–243 psychiatric disorders 241–247 signal quantiﬁcation techniques 43–45 water suppression techniques 42–43 proton leakage 120 proton observe carbon edit (POCE) method 49, 155–156 psychiatric disorders 13 C MRS 247–250 glutamate–glutamine-GABA cycles 243–248

Index 1 H MRS 241–247, 249 N-acetyl aspartate levels 242–243 neurotransmitter systems 239–251 pulsed NMR 32 pyramidal neurons 178–179 pyridoxine 227 pyruvate carboxylase 79, 82, 83

quantiﬁcation of brainwork 4–5 radioactive tracers 2-[14 C]deoxyglucose method 13–14, 18 cerebral blood ﬂow and metabolism studies 12 2-[18 F]deoxy-D-glucose method 14–15 isotope labelling data 53–72 PET study limitations x rats, anesthesia studies 15, 179–184, 284, 298, 301–302 relaxivity 130–131 relevance ratio 67–68 repetitive tasks 286–287 resting potential 113, 118 restless brain concept 299, 311 retrieval, long term memory 264, 268–269 scalar coupling 45–46, 47 scatter effects 64–66 schizophrenia 128, 244–245 sciatic nerve 20–21, 23 sensory cortex 303–304 shimming techniques 40–42 shunts, GABA 102–103 signal-to-noise ratio (SNR) 130–132 signalling 112–122 signiﬁcance thresholds 281 single cell recordings 177 single-unit activity (SUA) recording 177, 184 sleep deprivation 289 SNR see signal-to-noise ratio sodium ions see Na+ somatosensory cortex 178–179, 180–181 somatosensory processing 179–184, 284, 301–302 spatial localization techniques 39–40 spectral editing techniques 45–50 spiking frequency (ν) see also action potentials measuring changes 179–181

321 redeployment of distributions 186–187 relationship to CMRO2 181–184 stimulus induced incremental activity 299–301 striate cortex 20, 21 structure/function relationship (brain) 280 SUA see single-unit activity recording substrate sources 59 subtraction method 280–284 see also differencing methods synapses 20–21, 24, 113–115, 121 target data 60 TCA cycle see tricarboxylic acid cycle techniques in NMR 31–52 temporal lobe epilepsy (TLE) 198–199, 217–227, 230 tetrodes 191 thalamus 179 thermodynamic approach 295–297 tissue heterogeneity 199, 201–202, 205–208 TLE see temporal lobe epilepsy total brain activity 299, 303–306 see also baseline activity tracers see radioactive tracers tricarboxylic acid (TCA) cycle 13 C MRS measurements of cerebral energy metabolism 76, 78 ﬂuoroacetate inhibition 92 GABA synthesis 100, 102 glutamate–glutamine cycle rate ratios 222–225 modeling 54 tumors, glutamate release 219 two-compartment model 55, 68–69 two-dimensional NMR spectroscopy 45 ultrahigh ﬁeld 17 O NMR 130–132, 134–143 unconscious activity 305, 314 VAPRO signal quantiﬁcation algorithm 43 verbalization 266 vesicle release, glutamatergic synapses 114 visual occlusion 20, 21 voltage inside cells 174, 177 water 132–134 suppression 33, 42–43 white matter 92, 199–202 work (brainwork) 4–5, 185, 186–187, 190 writing systems 285

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